Cellulases, the genes encoding them and uses thereof

ABSTRACT

Genes encoding novel cellulases, and a gene encoding a protein that facilitates the action of such novel cellulases, the novel cellulases and a protein that facilitates the action of such cellulases, and enzyme preparations containing such proteins are described The native hosts and the culture medium of said hosts containing said novel cellulases are also disclosed. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

This application is a divisional of U.S. application Ser. No.08/841,636, filed Apr. 30, 1997 (pending), which is a continuation ofInternational Application No. PCT/FI96/00550, filed Oct. 17, 1996, and acontinuation-in-part of U.S. application Ser. No. 08/732,181, filed Oct.16, 1996 (abandoned), which claims the benefit of U.S. Provisionalapplication Ser. Nos. 60/005,335, filed Oct. 17, 1995; 60/007,926, filedDec. 4, 1995; and 60/020,840, filed Jun. 28, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to genes encoding novel neutralcellulases and compositions containing the novel neutral cellulases.These compositions are especially useful in the textile, detergent andpulp and paper industries.

2. Related Art

Cellulose is a linear polysaccharide of glucose residues connected byp-1,4 linkages. In nature, cellulose is usually associated with lignintogether with hemicelluloses such as xylans and glucomannans. Thepractical use of cellulases has been hampered by the nature of the knowncellulases, which are often mixtures of cellulases having a variety ofactivities and substrate specificities. For that reason, it is desirableto identify sources from which cellulases having only the desiredactivities may be obtained.

A wide variety of cellulases are known in the art, most of which areacid cellulases. However, some neutral and alkaline cellulases have alsobeen identified. Celluzyme® is a commercially-available cellulasepreparation from Humicola insolens (Novo Nordisk, A/S). GB 2,075,028 andEP 406,314 describe the use of a Humicola insolens cellulase as anenzymatic additive in a wash detergent to reduce the harshness(stiffness) of cotton-containing fabrics. The cloning of a cellulasecontaining endoglucanase activity from Humicola insolens is described inWO 93/11249 and EP 531,372. EP 510,091 describes a cellulase fromBacillus spp. NCI 40250 that is useful in detergent compositions. EP220,016 describes cellulases that are useful as clarification agents forcolored fabrics. WO 94/07998 describes modified cellulases that possessan improved alkaline activity. WO 95/02675 describes detergentcompositions that contain two different cellulases: a first cellulasethat is catalytically amenable to particulate soil removal, and a secondcellulase that is catalytically amenable to color clarification. WO921/8599 describes a detergent preparation that contains both acellulase and a protease. Cellulases have also been used industrially asan aid for the removal of printing paste thickener and excess dye aftertextile printing (EP 576,526).

EP 383 828 describes granular detergent compositions, which containsurface-active agent, a fabric-softening clay material, and cellulasegranulates containing calcium carbonate. U.S. Pat. No. 5,433,750describes detergent compositions containing a surface active agent, abuilder system, a softening clay, a clay flocculating agent and a highactivity cellulase, preferably Humicola insolens cellulase. U.S. Pat.No. 5,520,838 describes granular detergent compositions, comprisingsurface-active agent, a builder and a cellulase, preferably a Humicolainsolens cellulase, said compositions being in a compact form, having arelatively high density and containing a low amount of inorganic fillersalt.

Cellulase enzymes are used in a wide variety of industries in additionto the textile industry. For example, cellulases are used industriallyfor the deinking of newspapers and magazines (EP 521,999), for improvingthe drainage of pulp (WO 91/14822, WO 91/17243), and as a treatment foranimal feed.

The unique properties of each cellulase make some more suitable forcertain purposes than others. While the enzymes differ in a number ofways, one of the most important difference is pH optimum. Neutralcellulases have useful cellulase activity in the pH range 6-8, alkalinecellulases have useful cellulase activity in the pH range 7.5-10. Acidcellulases are active in the range of pH 4.5-6, but have littlecellulase activity at higher pH values.

Neutral and acid cellulases are especially useful in the textileindustry Klahorst, S. et at, Textile Chemist and Colorist 26:13-18,1994; Nilsson, T. E., Aachen Textile Conference, DWI Reports 114:85-88(1995); Videbaek, T. et al., ITB Dyeing/Printing/Finishing, January1994, pp. 25-29; Klahorst, S. et al., AATCC Int. Conf & Exhibit, October4-7, 1992, p. 243, Atlanta, Ga.; Kochavi, D. et al., Am. DyestuffResporter, September 1990, pp. 26-28; Tyndall, R. Michael, TextileChemist and Colorist 24:23 (1992); Lange, N. K., in Proc. Second TRICELSymp. on Trichoderma reesei Cellulases and Other Hydrolases, Espoo,Finland, 1993, ed. P. Suominen et al., Foundation for Biotechnical andIndustrial Fermentation Research vol. 8, 1993, pp. 263-272. When used totreat fabric, cellulases attack the chains of cellulose molecules thatform the cotton fibers, thereby affecting the characteristics of thefabric.

Traditionally, in “stonewashing,” pumice stones have been used to changethe characteristics of the fabric. Gradually, cellulases are replacingpumice stones, which also give the fabric its desired final look but cancause damage to the machines, garments and sewage processing equipment.U.S. Pat. No. 4,832,864, U.S. Pat. No. 4,912,056, U.S. Pat. No.5,006,126, U.S. Pat. No. 5,122,159, U.S. Pat. No. 5,213,581 and EP307,564 disclose the use of cellulases in biostoning.

Cellulases are especially useful for stonewashing denim dyed with indigoas the dye mostly stays on the surface of the yarn and does notpenetrate the fibers well. When used to treat cotton fabric, neutralcellulases generally require a longer wash time than do the acidcellulases. However, available neutral cellulases are less aggressive(active) against cotton than acid cellulases, and are reported not tocompromise the strength of the fabric as readily as acid cellulases.Neutral cellulases have a broader pH profile and thus the pH increasethat occurs during biostoning has little effect on the activity of theneutral enzyme.

The use of acid cellulases is hampered by their tendency to promotebackstaining and a weakening of fabrics. In addition, the pH must beadjusted to to a range suitable for the function of the acid cellulases.Consequently, there is a clear demand for neutral cellulase enzymepreparations that do not cause backstaining or weakening of fabrics.

While it has become popular to use cellulases in the textile industry,simply changing the cellulase mixture that is used may produce adifferent finish. These problems have focused increasing attention onthe search for reproducible mixtures of cellulases with desiredproperties. Thus there is a clear demand especially in the textile anddetergent industry for novel cellulases active at neutral and alkalinepH values, not compromising the strength of fabrics, with good cleaningand/or fabric care and harshness reducing properties.

SUMMARY OF THE INVENTION

Recognizing the importance of identifying enzymes useful in textilebiofinishing and biostoning and in detergent applications, the inventorshave screened fungal species for neutral and alkaline cellulases withenzymatic characteristics that would be useful in such technologies.

These studies have resulted in novel cellulases originating from thegenera Myceliophthora, Myriococcum, Melanocarpus, Sporotrichum andChaetomium.

The invention is further directed to the spent culture medium or enzymepreparations prepared from the native hosts producing such novelcellulases.

The invention is further directed to the use of such culture medium orthe use of such enzyme preparations in the textile and detergentindustry and in the pulp and paper industries.

These studies have further resulted in the identification of three novelcellulases that are especially useful in the textile and detergentindustry. Purified preparations from Melanocarpus sp. or Myriococcum sp.have revealed a 20 kDa cellulase with endoglucanase activity (designatedherein as “20K-cellulase”), a 50 kDa cellulase (“50K-cellulase”), and asecond 50 kDa cellulase (“50K-cellulase B”). A novel gene product withhigh homology to the cellulase family, herein called “protein-with-CBD”(where CBD means “cellulose binding domain”) was also discovered.

It is an object of the invention to provide enzyme preparations thatcontain one or more of the novel cellulases of the invention, especiallythe 20K-cellulase, the 50K-cellulase, the 50K-cellulase B and/or theprotein-with-CBD.

It is a further object of this invention to provide a method for usingsuch preparations for the finishing of textiles, especially thebiostoning of denim, for the use said preparations in detergentcompositions, and especially methods that use the 20K-cellulase, the50K-cellulase, the 50K-cellulase B and/or the protein-with-CBD.

The invention is also directed to other neutral and/or alkalinecellulases having one or more of the amino acid sequences as describedherein.

The invention is further directed to the genes encoding the20K-cellulase, 50K-cellulase, 50K-cellulase B and the protein-with-CBD.

The invention is further directed to novel expression vectors comprisingsuch genes and to novel hosts transformed with the vectors, especiallyhosts that are capable of high levels of expression of the proteinsencoded by such genes.

The invention is further directed to the spent culture medium of suchtransformed hosts, the culture medium containing the novel20K-cellulase, 50K-cellulase, the 50K-cellulase B and/or theprotein-with-CBD, or enzyme compositions (enzyme preparations)containing one or more of these proteins that have been prepared fromsuch culture media.

The invention is further directed to the use of such culture medium orthe use of such enzyme preparations in the textile and detergentindustry and in the pulp and paper industries.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1(A and B) show the pH (FIG. 1A) and temperature (FIG. 1B)dependencies of the endoglucanase activities of ALKO4179, CBS 689.95

FIGS. 2(A and B) show the pH (FIG. 2A) and temperature (FIG. 2B)dependencies of the endoglucanase activities of ALKO4124, CBS 687.95.

FIGS. 3(A and B) show the pH (FIG. 3A) and temperature (FIG. 3B)dependencies of the endoglucanase activities of ALKO4237, CBS 685.95.

FIGS. 4(A and B) show the pH FIG. 4A) and temperature (FIG. 4B)dependencies of the endoglucanase activities of ALKO4265, CBS 730.95.

FIGS. 5(A and B) show the pH (FIG. 5A) and temperature (FIG. 5B)dependencies of the endoglucanase activities of ALKO4125, CBS 688.95.

FIGS. 6(A and B) show the wash effect and backstaining (FIG. 6A) andblueness (FIG. 6B) with the neutral cellulases.

FIGS. 7(A and B) show the wash effect and backstaining (FIG. 7A) andblueness (FIG. 7B) with Ecostone L with gradually increasing enzymedosages. 1× corresponds the enzyme dosage of the neutral cellulases inFIGS. 6A and 6B.

FIG. 8 shows the purification of 20K-cellulase from Peak II bychromatography on SP-Sepharose™. A sample containing 11.7 g of proteinand 576,000 ECU was applied to a 4.5×31 cm column, which was developedas described in Example 9. Fractions of 15 ml were collected.Endoglucanase activities in the peak at fractions 148-161 areunderestimated because crystallization occurred before the enzyme couldbe sufficiently diluted for assay. Crystalline material from thesefractions contained 486,000 ECU.

FIGS. 9(A and B) show SDS-PAGE analysis of the 20K-cellulase. Themolecular masses of the standards are shown in kDa

A Partially crystalline material precipitated from the activeS-Sepharose™ fractions (lane 1).

B Fractions from chromatography of the partially crystalline material onG50 Sephadex. Fractions shown in lanes 19 and 25 contained noendoglucanase activity. For the other fractions, the amounts of activity(in ECU) applied to the gel was as follows: fraction 27, 0.4; 29, 2.4(as 3.0 μg of protein); 30, 2.1; 31, 1.9; 33, 0.46; and 35, 1.1.

FIG. 10 shows the separation of 50K-cellulase and 50K-cellulase B fromPeak III/IV by chromatography on SP-Sepharose™. A sample containing 200mg of protein and 14,800 ECU was applied to the 2.5×11 cm column, whichwas developed as described in Example 9. Fractions of 6.8 ml werecollected. A minor amount of 50K-cellulase eluted before the NaClgradient, whereas most of the 50K-cellulase eluted at about 50 mM NaCl.50K-cellulase B was found in the major protein peak at about 80 mM NaCl.

FIG. 11 shows an SDS-PAGE analysis of purified 50K-cellulase (11A) and50K-cellulase B (11B). Lane numbers indicate the fractions (3.3 ml)eluted from Phenyl-Sepharose. For fractions 36-41, 2.5 μl of eachfraction was applied to the gel. For the other fractions, 2 μl wasapplied. The 50K-cellulase peak was found in fractions 37-38 (11A)(containing 780 and 880 ECU/ml, respectively). The 50K-cellulase B peakwas in fractions 30 and 31 (11 B), which contained negligible activity(less than 4 ECU/ml).

FIG. 12 shows the temperature dependence of the endoglucanase activityof 50K-cellulase at pH 7.0 and a reaction time of 60 min.

FIG. 13 shows the pH dependence of the endoglucanase activity of50K-cellulase at 50° C. (♦) and 70° C. (□).

FIG. 14 shows a Western analysis using 20K-cellulase antiserum as aprobe. Lanes 1, 2 and 3 contain 25 μg of protein from the DEAE-Sepharosepeaks 1, III and IV, respectively. Lanes 4 and 5 contain 2.0 and 0.2 μgof pure 50K-cellulase and lane 6 contains 0.6 μg of pure 50K-cellulaseB. Lanes 7 and 8 contain about 25 μg protein from the whole growthmedium of ALKO4237 and ALKO4124, respectively.

FIG. 15 shows the temperature dependence of the endoglucanase activityof 20K-cellulase at pH 7 (10 min reaction times).

FIGS. 16(A and B) show the pH-dependence of the endoglucanase activityof the 20K-cellulase for the reaction time of (a) 10 minutes or (b) 60minutes.

FIG. 17 shows amino acid sequence data derived from sequencing the20K-cellulase described in the exemplary material herein. Sequence 429(SEQ ID NO:1) is from the N terminus of the protein and the othersequences are from internal tryptic peptides. Sequence #430 correspondsto SEQ ID NO: 2; sequence #431 corresponds to SEQ ID NO: 3; sequence#432 corresponds to SEC, ID NO: 4; sequence #433 corresponds to SEQ IDNO: 5; sequence #439 corresponds to SEQ ID NO: 6; fr 9 corresponds toSEQ ID NO: 7; fr 14 corresponds to SEQ ID NO: 8; fr 16 corresponds toSEQ ID NO: 9; fr 17 corresponds to SEQ ID NO: 10; fr 28 corresponds toSEQ ID NO: 11 and fr 30 corresponds to SEQ ID NO: 12.

FIG. 18 shows the restriction maps of the Melanocarpus albomyces DNA inplasmids pALK1221, pALK1222 and pALK1223, which carry the 20K-cellulasegene.

FIG. 19(A and B) shows the DNA sequence of the 20K-cellulase gene (SEQID NO:30). The arrow indicates the predicted signal peptidase processingsite.

FIG. 20 shows the restriction maps of the Melanocarpus albomyces DNA inplasmids pALK1233, pALK1234, pALK1226 and pALK1227, which carry the50K-cellulase gene.

FIGS. 21(A, B and C) show the DNA sequence of the 50K-cellulase gene(SEQ ID NO:32). The arrow indicates the predicted signal peptidaseprocessing site.

FIG. 22 shows the restriction maps of the Melanocarpus albomyces DNA inplasmids pALK1229 and pALK1236, which carry the 50K-cellulase B gene.

FIGS. 23(A, B and C) show the DNA sequence of the 50K-cellulase B gene(SEQ ID NO:34). The arrow indicates the predicted signal peptidaseprocessing site.

FIG. 24 shows the plasmid map of pTTc01.

FIG. 25 shows the plasmid map of pMS2.

FIG. 26 shows the restiction map of the Melanocarpus albomyces DNA inplasmid pALK1230, which carries DNA encoding the protein-with-CBD. Thesequence presented in FIG. 27 (SEQ ID NO:36) is marked with an arrow inFIG. 26.

FIG. 27 shows the DNA sequence of the the protein-with-CBD cellulasegene (SEQ ID NO:36) in pALK1230.

FIG. 28 shows the plasmid map of pALK1231.

FIG. 29 shows the plasmid map of pALK1235.

FIG. 30 shows a Western analysis using 20K-cellulase antiserum as aprobe. Lanes 1 and 2 contain about 10 μg protein from the whole growthmedium of transformants ALKO3620/pALK1235/49 and ALKO3620/pALK1235/40.Lane 3 contains about 10 μg protein from the whole growth medium ofALKO3620. Lanes 4 and 5 contain about 10 μg protein from the wholegrowth medium of transformants ALKO3620/pALK1231/16 andALKO3620/pALK1231/14. Lane 6 contains 100 ng of pure 20K-cellulase.

FIG. 31 shows the plasmid map of pALK1238.

FIG. 32 shows the plasmid map of pALK1240.

DEPOSITS

ALKO4179, Myceliophthora thermophila was deposited as CBS 689.95 on Oct.12, 1995, at the Centraalbureau voor Schimmelcultures, P.O. Box 273,3740AG BAARN.

ALKO4124, Myriococcum sp. was deposited as CBS 687.95 on Oct. 12, 1995,at the Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AGBAARN.

ALKO4237, Melanocarpus albomyces (=Myriococcum albomyces=Thielaviaalbomyces; Guarro et al., 1996, Mycol. Res. 100(1):75.) was deposited asCBS 685.95 on October 11, 1995, at the Centraalbureau voorSchimmelcultures, P.O. Box 273, 3740 AG BAARN.

ALKO4125, Sporotrichum thermophile was deposited as CBS 688.95 on Oct.12, 1995, at the Centraalbureau voor Schimmelcultures, P.O. Box 273,3740 AG BAARN.

ALKO4265, Chaetomium thermophilum La Touche was deposited as CBS 730.95on Nov. 8, 1995, at the Centraalbureau voor Schimmelcultures, P.O. Box273,3740 AG BAARN.

Plasmid pALK1221 was deposited as DSM 11024 on Jun. 21, 1996 andλ423715.1 was deposited as DSM 11012 on Jun. 21, 1996, at the DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B,D-38124 Braunschweig, Germany. Both contain the 20K-cellulase gene fromMelanocarpus albomyces CBS 685.95.

Plasmid pALK1227 was deposited as DSM 11025 on Jun. 21, 1996 andλ4237/35 was deposited as DSM 11014 on Jun. 21, 1996, at the DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B,D-38124 Braunschweig, Germany. Both contain the 50K-cellulase gene fromMelanocarpus albomyces CBS 685.95.

Plasmid pALK1229 was deposited as DSM 11026 on Jun. 21, 1996 and λ4237/3was deposited as DSM 11011 on Jun. 21, 1996, and λ4237/18 was depositedas DSM 11013 on Jun. 21, 1996, at the Deutsche Sammulung vonMikroorganismen und Zelikulturen GmbH, Mascheroder Weg 1B, D-38124Braunschweig, Germany. pALK1229 contains DNA coding for the50K-cellulase B, λ4237/3 and A4237/18 contain the 50K-cellulase B genefrom Melanocarpus albomyces CBS 685.95.

Plasmid pALK1230 was deposited as DSM 11027 on Jun. 21, 1996 at theDeutsche Sammulung von Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1B, D-38124 Braunschweig, Germany. pALK1230 contains theprotein-with-CBD gene from Melanocarpus albomyces CBS 685.95.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the description that follows, a number of terms used in textileindustry technology are extensively utilized. In order to provide aclear and consistent understanding of the specification and claims,including the scope to be given such terms, the following definitionsare provided.

Biostoning. “Biostoning” of fabric or garment means the use of enzymesin place of, or in addition to, the use of pumice stones for thetreatment of fabric or garment, especially denim.

Biofinishing. “Biofinishing” refers to the use of enzymes in acontrolled hydrolysis of cellulosic fibers in order to modify the fabricor yam surface in a manner that prevents permanently pilling, improvesfabric handle like softness and smoothness, clears the surface structureby reducing fuzzing, which results in clarification of colours, improvesthe drapability of the fabric, improves moisture absorbability and whichmay improve also the dyeability.

Backstaining. Released dye has a tendency to redeposit on the surface ofthe fabric fibers. This effect is termed “backstaining.”

Detergent. By “detergent” is meant a cleansing agent that can containsurface active agents (anionic, non-ionic, cationic and ampholyticsurfactants), builders and other optional ingredients such asantiredeposition and soil suspension agents, optical brighteners,bleaching agents, dyes and pigments and hydrolases. Suitable listing ofthe contents of detergents is given in U.S. Pat. No. 5,433,750, asuitable list of surfactants is given in U.S. Pat. No. 3,664,961.

Enzyme preparation. By “enzyme preparation” is meant a compositioncontaining enzymes. Preferably, the enzymes have been extracted from(either partially or completely purified from) a microbe or the mediumused to grow such microbe. “Extracted from” means that the desiredenzymes are separated from the cellular mass. This can be performed byany method that achieves this goal, including breaking cells and alsosimply removing the culture medium from spent cells. Therefore, the term“enzyme preparation” includes compositions containing medium previouslyused to culture a desired microbe(s) and any enzymes that have beenreleased from the microbial cells into such medium during the culture ordownstream processing steps.

By a host that is “substantially incapable” of synthesizing one or moreenzymes is meant a host in which the activity of one or more of thelisted enzymes is depressed, deficient, or absent when compared to thewild-type.

By an amino acid sequence that is an “equivalent” of a specific aminoacid sequence is meant an amino acid sequence that is not identical tothe specific amino acid sequence, but rather contains at least someamino acid changes (deletions, substitutions, inversions, insertions,etc) that do not essentially affect the biological activity of theprotein as compared to a similar activity of the specific amino acidsequence, when used for a desired purpose. The biological activity of acellulase, is its catalytic activity, and/or its ability to bind tocellulosic material. The biological activity of the 50K-cellulase Bfurther includes its ability to act synergistically with the cellulases.Preferably, an “equivalent” amino acid sequence contains at least80%-99% identity at the amino acid level to the specific amino acidsequence, most preferably at least 90% and in an especially highlypreferable embodiment, at least 95% identify, at the amino acid level.

Cloning vehicle. A cloning vehicle is a plasmid or phage DNA or otherDNA sequence (such as a linear DNA) that provides an appropriate nucleicacid carrier environment for the transfer of a gene of interest into ahost cell. The cloning vehicles of the invention may be designed toreplicate autonomously in prokaryotic and eukaryotic hosts. In fungalhosts such as Trichoderma, the cloning vehicles generally do notautonomously replicate and instead, merely provide a vehicle for thetransport of the gene of interest into the Trichoderma host forsubsequent insertion into the Trichoderma genome. The cloning vehiclemay be further characterized by one or a small number of endonucleaserecognition sites at which such DNA sequences may be cut in adeterminable fashion without loss of an essential biological function ofthe vehicle, and into which DNA may be spliced in order to bring aboutreplication and cloning of such DNA. The cloning vehicle may furthercontain a marker suitable for use in the identification of cellstransformed with the cloning vehicle. Markers, for example, areantibiotic resistance. Alternatively, such markers may be provided on acloning vehicle which is separate from that supplying the gene ofinterest. The word “vector” is sometimes used for “cloning vehicle.”

Expression vehicle. An expression vehicle is a cloning vehicle or vectorsimilar to a cloning vehicle but which is capable of expressing a geneof interest, after transformation into a desired host. When a fungalhost is used, the gene of interest is preferably provided to a fungalhost as part of a cloning or expression vehicle that integrates into thefungal chromosome, or allows the gene of interest to integrate into thehost chromosome. Sequences that are part of the cloning vehicle orexpression vehicle may also be integrated with the gene of interestduring the integration process. In T. reesei, sites of integration towhich the gene of interest can be directed include the cbh and/or theegl loci. Most preferably, the gene of interest is directed to replaceone or more genes encoding undesirable characteristics.

The gene of interest is also preferably placed under the control of(i.e., operably linked to) certain control sequences such as promotersequences provided by the vector (which integrate with the gene ofinterest). Alternatively, the control sequences can be those at theinsertion site.

The expression control sequences of an expression vector will varydepending on whether the vector is designed to express a certain gene ina prokaryotic or in a eukaryotic host (for example, a shuttle vector mayprovide a gene for selection in bacterial hosts). Expression controlsequences can contain transcriptional regulatory elements such as,promoters, enhancer elements, and transcriptional termination sequences,and/or translational regulatory elements, such as, for example,translational initiation and termination sites.

According to the invention, there are provided neutral and alkalinecellulases, and methods for producing such useful neutral and alkalinecellulases, that are desirable for the treatment of textile materials.

The native hosts that produce the proteins of the invention are:

1) ALKO4179, Myceliophthora thermophila; deposited as CBS 689.95 at theCentraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG BAARN.

2) ALKO4124, Myriococcum sp.; deposited as CBS 687.95;

3) ALKO4237, Melanocarpus albomyces, deposited as CBS 685.95;

4) ALKO4125, Sporotrichum thermophila, deposited as CBS 688.95; and

5) ALKO4265, Chaetomium thermophilum La Touche, deposited as CBS 730.95

One specific preferred embodiment of the invention is the spent culturemedium of the native hosts or enzyme preparations prepared from theculture medium.

In specific preferred embodiments of the invention, the purified20K-cellulase, 50K-cellulase, 50K-cellulase B and/or protein-with-CBD isprovided. These proteins can be obtained for example from Melanocarpussp. or Myriococcum sp. as described herein, and especially in Example 9.

Amino acid sequence data have been generated from the cellulasesdescribed herein. Accordingly, the invention is also directed to neutralor alkaline cellulases containing one or more of the amino acidsequences shown herein. Thus, the invention is intended to be directedto any neutral or alkaline cellulase that is a functional equivalent ofthe 20K-cellulase, the 50K-cellulase, the 50K-cellulase B and/orprotein-with-CBD and having one or more of the amino acid sequencesdescribed herein, or substantially the same sequence. Such neutral oralkaline cellulases can be derived from other strains of the samespecies or from divergent organisms.

In further preferred embodiments, the 20K-cellulase is provided with thematerial from separate peaks formed during the exemplified purificationprocedures (e.g., DEAE-Sepharose Pools I, III, or IV in Table VIIIherein). In still furhter embodiments, other proteins in theMelanocarpus albomyces ALKO 4237 medium may be used, either alone or incombination with other such proteins.

In further preferred embodiments, the 50K-cellulase is provided with thematerial from separate peaks formed during the exemplified purificationprocedures. In still further embodiments, other proteins in the ALKO4237 medium may be used, either alone or in combination with other suchproteins.

In further preferred embodiments, the 50K-cellulase B is provided withthe material from separate peaks formed during the exemplifiedpurification procedures. In still further embodiments, other proteins inthe ALKO 4237 medium may be used, either alone or in combination withother such proteins.

As described herein, ALKO 4265, Chaetomium thermophilum La Touche,deposited as CBS 730.95, is used herein as an example of a neutralcellulase that is not preferred in biostoning method of the inventionbecause it causes backstaining. However, there is evidence that it isuseful in other applications (e.g. in detergents).

The process for genetically engineering the hosts of the invention isfacilitated through the cloning of genetic sequences that encode thedesired protein and through the expression of such genetic sequences. Asused herein the term “genetic sequences” is intended to refer to anucleic acid molecule (preferably DNA). Genetic sequences that encodethe desired protein are derived from a variety of sources. These sourcesinclude genomic DNA, cDNA, synthetic DNA and combinations thereof.Vector systems may be used to produce hosts for the production of theenzyme preparations of the invention. Such vector construction (a) mayfurther provide a separate vector construction (b) which encodes atleast one desired gene to be integrated to the genome of the host and(c) a selectable marker coupled to (a) or (b). Alternatively, a separatevector may be used for the marker.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains expression control sequenceswhich contain transcriptional regulatory information and such sequencesare “operably linked” to the nucleotide sequence which encodes thepolypeptide.

An operable linkage is a linkage in which a sequence is connected to aregulatory sequence (or sequences) in such a way as to place expressionof the sequence under the influence or control of the regulatorysequence. Two DNA sequences (such as a protein encoding sequence and apromoter region sequence linked to the 5′ end of the encoding sequence)are said to be operably linked if induction of promoter function resultsin the transcription of the protein encoding sequence mRNA and if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the expression regulatory sequences to direct the expressionof the mRNA, antisense RNA, or protein, or (3) interfere with theability of the template to be transcribed by the promoter regionsequence. Thus, a promoter region would be operably linked to a DNAsequence if the promoter were capable of effecting transcription of thatDNA sequence.

The precise nature of the regulatory regions needed for gene expressionmay vary between species or cell types, but shall in general include, asnecessary, 5′ non-transcribing and 5′ non-translating (non-coding)sequences involved with initiation of transcription and translationrespectively. Expression of the protein in the transformed hostsrequires the use of regulatory regions functional in such hosts. A widevariety of transcriptional and translational regulatory sequences can beemployed. In eukaryotes, where transcription is not linked totranslation, such control regions may or may not provide an initiatormethionine (AUG) codon, depending on whether the cloned sequencecontains such a methionine. Such regions will, in general, include apromoter region sufficient to direct the initiation of RNA synthesis inthe host cell.

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the protein, or a functional derivativethereof, does not contain any intervening codons which are capable ofencoding a methionine. The presence of such codons results either in aformation of a fusion protein (if the AUG codon is in the same readingframe as the protein encoding DNA sequence) or a frame-shift mutation(if the AUG codon is not in the same reading frame as the proteinencoding sequence).

In a preferred embodiment, a desired protein is secreted into thesurrounding medium due to the presence of a secretion signal sequence.If a desired protein does not possess its own signal sequence, or ifsuch signal sequence does not function well in the host, then theprotein's coding sequence may be operably linked to a signal sequencehomologous or heterologous to the host. The desired coding sequence maybe linked to any signal sequence which will allow secretion of theprotein from the host. Such signal sequences may be designed with orwithout specific protease sites such that the signal peptide sequence isamenable to subsequent removal. Alternatively, a host that leaks theprotein into the medium may be used, for example a host with a mutationin its membrane.

If desired, the non-transcribed and/or non-translated regions 3′ to thesequence coding for a protein can be obtained by the above-describedcloning methods. The 3′-non-transcribed region may be retained for itstanscriptional termination regulatory sequence elements; the3-non-translated region may be retained for its translationaltermination regulatory sequence elements, or for those elements whichdirect polyadenylation in eukaryotic cells.

The vectors of the invention may further comprise other operably linkedregulatory elements such as enhancer sequences.

In a preferred embodiment, genetically stable transformants areconstructed whereby a desired protein's DNA is integrated into the hostchromosome. The coding sequence for the desired protein may be from anysource. Such integration may occur de novo within the cell or, in a mostpreferred embodiment, be assisted by transformation with a vector whichfunctionally inserts itself into the host chromosome, for example, DNAelements which promote integration of DNA sequences in chromosomes.

Cells that have stably integrated the introduced DNA into theirchromosomes are selected by also introducing one or more markers whichallow for selection of host cells which contain the expression vector inthe chromosome, for example the marker may provide biocide resistance,e.g., resistance to antibiotics, or heavy metals, such as copper, or thelike. The selectable marker gene can either be directly linked to theDNA gene sequences to be expressed, or introduced into the same cell byco-transformation.

Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to‘shuttle’ the vector between host cells of different species.

Once the vector or DNA sequence containing the construct(s) is preparedfor expression, the DNA construct(s) is introduced into an appropriatehost cell by any of a variety of suitable means, includingtransformation as described above. After the introduction of the vector,recipient cells are grown in a selective medium, which selects for thegrowth of transformed cells. Expression of the cloned gene sequence(s)results in the production of the desired protein, or in the productionof a fragment of this protein. This expression can take place in acontinuous manner in the transformed cells, or in a controlled manner.

Accordingly, the protein encoding sequences described herein may beoperably lied to any desired vector and transformed into a selectedhost, so as to provide for expression of such proteins in that host.

The subject matter of the invention are also nucleic acid moleculescoding for proteins having the biological activity of a cellulase andthat hybridize to any of the nucleic acid molecules described above orwhich are defined in the following:

A nucleic acid molecule encoding a polypeptide having the enzymaticactivity of a cellulase, selected from the group consisting of:

(a) nucleic acid molecules encoding a polypeptide comprising the aminoacid sequence as depicted in FIG. 19 (SEQ ID NO:31) or 21 (SEQ IDNO:33);

(b) nucleic acid molecules encoding a polypeptide comprising the aminoacid sequence as depicted in FIG. 23 (SEQ ID NO:35) or 27 (SEQ IDNO:37);

(c) nucleic acid molecules comprising the coding sequence of thenucleotide sequence as depicted in FIG. 19 (SEQ ID NO:30) or 21 (SEQ IDNO:32);

(d) nucleic acid molecules comprising the coding sequence of thenucleotide sequence as depicted in FIG. 23 (SEQ ID NO:34) or 27 (SEQ IDNO:36);

(e) nucleic acid molecules encoding a polypeptide comprising the aminoacid sequence encoded by the DNA insert contained in DSM 11024, DSM11012, DSM 11025 or DSM 11014;

(f) nucleic acid molecules encoding a polypeptide comprising the aminoacid sequence encoded by the DNA insert contained in DSM 11026, DSM11011, DSM 11013 or DSM 11027;

(g) nucleic acid molecules comprising the coding sequence of the DNAinsert contained in DSM 11024, DSM 11012, DSM 11025 or DSM 11014;

(h) nucleic acid molecules comprising the coding sequence of the DNAinsert contained in DSM 11026, DSM 11011, DSM 11013 or DSM 11027;

(i) nucleic acid molecules hybridizing to a molecule of any one of (a),(c), (e) or (g); and

(j) nucleic acid molecules the coding sequence of which differs from thecoding sequence of a nucleic acid molecule of any one of (a) to (i) dueto the degeneracy of the genetic code.

(k) nucleic acid molecules encoding a polypeptide having cellulaseactivity and having an amino acid sequence which shows at least 80%identity to a sequence as depicted in FIG. 19 (SEQ ID NO:31), 21 (SEQ IDNO:33), 23 (SEQ ID NO:35) or 27 (SEQ ID NO:37).

The term “hybridization” in this context means hybridization underconventional hybridization conditions, preferably under stringentconditions such as described by, e.g. Sambrook et al. (1989, MolecularCloning, A Laboratory Manual 2nd Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). These nucleic acid molecules thathybridize to the nucleic acid molecules according to the presentinvention in principle can be derived from any organism possessing suchnucleic acid molecules. Preferably, they are derived from fungi, namelyfrom those of the genera Melanocarpus, Myriococcum, Sporotrichum,Myceliophthora and Chaetonium. Nucleic acid molecules hybridizing to thenucleic acid molecules of the present invention can be isolated, e.g.,from genomic libraries or cDNA libraries of various organisms, namelyfungi.

Such nucleic acid molecules can be identified and isolated by using thenucleic acid molecules of the present invention or fragments of thesemolecules or the reverse complements of these molecules, e.g. byhybridization according to standard techniques (see Sambrook et al.(1989)).

As hybridization probe, e.g. nucleic acid molecules can be used thathave exactly or substantially the same nucleotide sequence indicated inthe Figures or fragments of said sequence. The fragments used ashybridization probes can also be synthetic fragments obtained byconventional synthesis techniques and the sequence of which issubstantially identical to that of the nucleic acid molecules accordingto the invention. Once genes hybridizing to the nucleic acid moleculesof the invention have been identified and isolated it is necessary todetermine the sequence and to analyze the properties of the proteinscoded for by said sequence.

The term “hybridizing DNA molecule” includes fragments, derivatives andallelic variants of the above-described nucleic acid molecules that codefor the above-described protein or a biologically active fragmentthereof. Fragments are understood to be parts of nucleic acid moleculeslong enough to code for the described protein or a biologically activefragment thereof. The term “derivative” means in this context that thenucleotide sequences of these molecules differ from the sequences of theabove-described nucleic acid molecules in one or more positions and arehighly homologous to said sequence. Homology is understood to refer to asequence identity of at least 40%, particularly an identity of atleast.60%, preferably more than 80% and still more preferably more than90%. The deviations from the nucleic acid molecules described above canbe the result of deletion, substitution, insertion, addition orcombination.

Homology furthermore means that the respective nucleotide sequences orencoded proteins are functionally and/or structurally equivalent. Thenucleic acid molecules that are homologous to the nucleic acid moleculesdescribed above and that are derivatives of said nucleic acid moleculesare regularly variations of said molecules which represent modificationshaving the same biological function. They may be naturally occurringvariations, such as sequences of other organisms or mutations. Thesemutations may occur naturally or may be achieved by specificmutagenesis. Furthermore, these variations may be synthetically producedsequences. The allelic variants may be naturally occurring variants aswell as synthetically produced or genetically engineered variants.

The proteins encoded by the various variants of the nucleic acidmolecules of the invention share specific common characteristics, suchas enzymatic activity, molecular weight, immunological reactivity,conformation, etc., as well as physical properties, such aselectrophoretic mobility, chromatographic behaviour, sedimentationcoefficients, solubility, spectroscopic properties, stability, pHoptimum, temperature optimum, etc. Enzymatic activity of the cellulasecan be detected e.g. as described on page 11 and in Examples 1 and 25.

The present invention furthermore relates to nucleic acid molecules thesequences of which differ from the sequences of the above-identifiedmolecules due to degeneracy of the genetic code, and which code for aprotein having the biological activity of a cellulase.

The nucleic acid molecules of the invention are preferably RNA or DNAmolecules, most preferably genomic DNA or cDNA.

The present invention also relates to antibodies which specificallyrecognize one of the above-described proteins according to the inventionas well as to antibody fragments which have this property. Theseantibodies may be monoclonal or polyclonal. Methods for their productionare well known in the art and are described in detail, for example, inHarlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, ColdSpring Harbor Laboratory (1988).

Furthermore, the present invention relates to oligonucleotides whichspecifically hybridize with a nucleic acid molecule according to theinvention or with the complementary strand of such a nucleic acidmolecule. In this respect the term “specifically hybridize” means thatsuch an oligonucleotide hybridizes under stringent hybridizationconditions specifically to a nucleic acid molecule of the invention anddoes not show under such conditions cross-hybridization with sequencescoding for other polypeptides. Preferably such oligonucleotides have alength of at least 10 nucleotides, more preferably of at least 15nucleotides and most preferably of at least 30 nucleotides. They arepreferably no longer than 100 nucleotides, more preferably no longerthan 80 nucleotides and most preferably no longer than 60 nucleotides.In order to ensure that they specifically hybridize to a nucleic acidmolecule of the present invention such oligonucleotides show over theirtotal length an identity of at least 80%, preferably of at least 95% andmost preferably of at least 99% with a corresponding nucleotide sequenceof a nucleic acid molecule of the present invention. Theseoligonucleotides may be used, e.g., as probes for screening forsequences encoding cellulases in genomic or cDNA libraries or as PCRprimers.

The protein encoding sequences described herein may be fused in frame toother sequences so as to construct DNA encoding a fusion protein. Forexample, a recombinant vector encoding a 50K-cellulase, a 20K-cellulase,a 50K-cellulase B or the protein-with-CBD gene can be prepared as above,except that the protein encoding sequence is fused with the sequence ofa T. reesei cellulase, hemicellulase or mannanase, or at least onefunctional domain of such cellulase, hemicellulase, or mannanase asdescribed in U.S. Pat. No. 5,298,405, WO 93/24622 and in GenBanksubmission L25310, each incorporated herein by reference. Especially,the cellulase, hemicellulase, or mannanase is selected from the groupconsisting of CBHI, CBHII, EGI, EGII, XYLI, XYLII and MANI, or a domainthereof, such as the secretion signal or the core sequence. Mannanasehas the same domain structure as that of the cellulases: a core domain,containing the active site, a hinge domain containing a serine-threoninerich region, and a tail, containing the binding domain.

Fusion peptides can be constructed that contain a mannanase orcellobiohydrolase or endoglucanase or xylanase core domain or the coreand the hinge domains from the same, fused to the desired proteinencoding sequence of the invention. The result is a protein thatcontains mannanase or cellobiohydrolase or endoglucanase or xylanasecore or core and hinge regions, and a 50K-cellulase, 20Kellulase,50K-cellulase B or the protein-with-CBD sequence. The fusion proteincontains both the mannanase or cellobiohydrolase or endoglucanase orxylanase, and the 50K-cellulase, 20K-cellulase, 50K-cellulase B or theprotein-with-CBD activities of the various domains as provided in thefusion construct.

Fusion proteins can also be constructed such that the mannanase orcellobiohydrolase or endoglucanase or xylanase tail or a desiredfragment thereof, is included, placed before the 50K-cellulase,20K-cellulase, 50K-cellulase B or the protein-with-CBD sequence,especially so as to allow use of a nonspecific protease site in the tailas a protease site for the recovery of the 50K-cellulase, 20K-cellulase,50K-cellulase B or the protein-with-CBD sequence from the expressedfusion protein. Alternatively, fusion proteins can be constructed thatprovide for a protease site in a linker that is placed before the50K-cellulase, 20K-cellulase, 50K-cellulase B or the protein-with-CBDsequence, with or without tail sequences.

New properties for the 20K- and 50K-cellulases and for the 50K-cellulaseB can be created by fusing domains, such as a cellulose binding domain(CBD), preferably with its linker, to the proteins of the invention.Preferably, such CBD's and linkers are the corresponding CBD and linkerdomains of a Trichoderma cellulase, mannanase or of the Melanocarpusalbomyces protein-with-CBD.

The invention provides methods for producing enzyme preparations thatare partially or completely deficient in an undesirable cellulolyticactivity (that is, in the ability to degrade cellulose) and enriched inthe 50K-cellulase, 20K-cellulase, 50K-cellulase B or theprotein-with-CBD protein, as desired for the textile or detergentindustry or for pulp and paper processing. By “deficient in cellulolyticactivity” is meant a reduced, lowered, or repressed capacity to degradecellulose to smaller oligosaccharides. Such cellulolytic activitydeficient preparations, and the making of same by recombinant DNAmethods, are described in U.S. Pat. No. 5,298,405, incorporated hereinby reference. Preferably, the preparation is deficient in EG activities,and/or CBHI activity.

As described herein, the 50K-cellulase, 20K-cellulase, 50K-cellulase Bor the protein-with-CBD may be provided directly by the hosts of theinvention. Alternatively, spent medium from the growth of the hosts, orpurified 50Kellulase, 20K-cellulase, 50K-cellulase B or theprotein-with-CBD therefrom, can be used. Further, if desired activitiesare present in more than one recombinant host, such preparations may beisolated from the appropriate hosts and combined prior to use in themethod of the invention.

To obtain the enzyme preparations of the invention, the native orrecombinant hosts described above having the desired properties (thatis, hosts capable of expressing economically feasible quantities of thedesired 50K-cellulase, 20Kellulase, 50K-cellulase B or protein-with-CBD,and optionally, those that are substantially incapable of expressing oneor more other, undesired cellulase enzymes) are cultivated undersuitable conditions, the desired enzymes are secreted from the hostsinto the culture medium, and the enzyme preparation is recovered fromsaid culture medium by methods known in the art.

The enzyme preparations of the invention can be produced by cultivatingthe recombinant hosts or native strains in a fermentor on a suitablegrowth medium (such as, for example, shown in Example 1 or in Example30).

The enzyme preparation can be the culture medium with or without thenative or transformed host cells, or is recovered from the same by theapplication of methods well known in the art. However, because the50K-cellulase, 20K-cellulase or 50K-cellulase B are secreted into theculture media and display activity in the ambient conditions of thecellulolytic liquor, it is an advantage of the invention that the enzymepreparations of the invention may be utilized directly from the culturemedium with no further purification. If desired, such preparations maybe lyophilized or the enzymatic activity otherwise concentrated and/orstabilized for storage. The enzyme preparations of the invention arevery economical to provide and use because (1) the enzymes may be usedin a crude form; isolation of a specific enzyme from the culture mediumis unnecessary and (2) because the enzymes are secreted into the culturemedium, only the culture medium need be recovered to obtain the desiredenzyme preparation; there is no need to extract an enzyme from thehosts. Preferably the host for such production is Trichoderma, andespecially T. reesei.

The enzyme preparations of the invention may be provided as a liquid oras a solid, for example, in a dried powder or granular or liquid form,especially nondusting granules, or a stabilized liquid, or the enzymepreparation may be otherwise concentrated or stabilized for storage oruse. It is envisioned that enzyme preparations containing one or more ofthe neutral cellulases of the invention can be further enriched or madepartially or completely deficient in specific enzymatic activities, soas to satisfy the requirements of a specific utility in variousapplications e.g. in the textile industry. A mixture of enzymeactivities secreted by a host and especially a fungus, can be chosen tobe advantageous in a particular industrial application, for examplebiostoning.

The enzyme preparations of the invention can be adjusted to satisfy therequirements of specific needs in various applications in the textile,detergent or the pulp and paper industry.

Blends may be prepared with other macromolecules that are not allsecreted from the same host (for example, other enzymes such asendoglucanases, proteases, lipases, peroxidases, oxidases or amylases)or chemicals that may enhance the performance, stability, or bufferingof the desired enzyme preparation. Non-dusting granules may be coated.Liquid enzyme preparations can be stabilized by adding a polyol such aspropylene glycol, a sugar or sugar alcohol, lactic acid or boric acid,according to established methods. Liquid detergents generally contain upto 90% water and 0-20% organic solvent. Protected forms of the enzymesof the invention may be prepared as described in EP 238,216.

The enzyme preparations of the invention can contain a surfactant whichcan be anionic, non-ionic, cationic, amphoteric or a mixture of thesetypes, especially when used as a detergent composition,. Usefuldetergent compositions are described e.g. in WO 94/07998, U.S. Pat. No.5,443,750 and U.S. Pat. No. 3,664,961.

If required, a desired enzyme may be further purified in accordance withconventional conditions, such as extraction, precipitation,chromatography, affinity chromatography, electrophoresis, or the like.

The enzyme preparations of this invention are especially useful intextile industry preferably in biostoning and in biofinishing or indetergent industry. Other useful areas are in pulp and paper industry.

Non-enzymatic stonewashing has three steps: desizing, abrasion andaftertreatment. The first step, desizing, involves the removal of thestarch coating, or that of its derivatives, by amylase. The second step,abrasion, when performed without cellulase, is generally performed bywashing the denim with pumice stones, and, when lightening is desired,bleach. The abrasive effect is the result not only of the effect of thestones but also the rubbing together of the denim fabric. Abrasion isgenerally followed by the third step, a washing step to remove excessdye, during which softeners or optical brighteners can be added.

In enzymatic stonewashing, or biostoning, abrasion with pumice stones iscompletely or partially eliminated and cellulase is added to facilitatethe abrasion of indigo dye from the fiber surface. After this treatment,the cellulase is removed with a detergent wash to ensure that themechanical strength of the fiber is not further compromised by thecontinued presence of the enzyme. Treatment with a cellulase(s) cancompletely replace treatment with pumice stones (for example, 1 kgcommercial enzyme per 100 kg stones). However, cellulase treatment canbe combined with pumice stone treatment when it is desired to produce aheavily abraded finish. A peach skin effect in which a fine protrudinghair-like covering is created is also achieved by a wash combining aneutral cellulase with pumice stones. The cellulases of this inventionare useful especially to minimize backstaining and enhance lightening(abrasion) in biostoning.

Biostoning is preferably performed from about pH 4.5-9.5, and mostpreferably between pH 6.0-8.5. The temperature of the reaction can rangefrom about 40-80° C., preferably between 50-70° C., and most preferablybetween 50-60° C. The liquid ratio (the ratio of the volume of liquidper weight of fabric) may range from about 2:1-20:1, preferably4:1-10:1, and most preferably 4:1-7:1. The enzyme dosage can range fromabout 25-1500 nkat/g fabric, preferably 50-500 nkat/g fabric and mostpreferably 75-300 nkat/g fabric.

The cellulases of the invention are useful in the textile industry forbiofinishing of fabrics or garments e.g. depilling, defuzzing, colourclarification, harshness reduction, the creation of different finishes(for example, a ‘peach skin,’ ‘worn out,’ ‘sand washed,’ or ‘antiquelook’ effect) and biofinishing of yarn (for example reduction ofhairiness, improvement of smoothness). The cellulases of this inventioncan be used in biofinishing in acidic and in neutral conditions.

The cellulases of this invention are useful in detergent compositions toimprove the textile cleaning effect e.g. soil removal, to improve thefabric-care properties by reducing the harshness of the textiles, thecellulases having also defuzzing and colour clarification and restoringeffects.

The textile material that is treated with the enzyme preparations of theinvention may be manufactured of natural cellulose containing fibers ormanmade cellulose containing fibers or mixtures thereof. Examples ofnatural cellulosics are cotton, linen, hemp, jute and ramie. Examples ofmanmade cellulosics are viscose, cellulose acetate, cellulosetriacetate, rayon, cupro and lyocell. The above mentioned cellulosicscan also be employed as blends of synthetic fibers such as polyester,polyamide or acrylic fibers. The textile material may be yarn or knittedor woven or formed by any other means.

The cellulases of the invention, besides being especially useful for thetreatment of fabric, are useful in general in any area requiringcellulase activity. In the pulp and paper industry, neutral cellulasescan be used, for example, in deinking of different recycled papers andpaperboards having neutral or alkaline pH, in improving the fiberquality, or increasing the drainage in paper manufacture. Other examplesinclude the removal of printing paste thickener and excess dye aftertextile printing, and as a treatment for animal feed. For example, ifthe intended application is improvement of the strength of themechanical pulp, then the 50K-cellulase, 20Kellulase, 50K-cellulase B orthe protein-with-CBD preparations of the invention may provide one ormore of these proteins so as to enhance or facilitate the ability ofcellulose fibers to bind together. In a similar manner, in theapplication of pulp refining, the 50K-cellulase, 20K-cellulase,50K-cellulase B or protein-with-CBD preparations of the invention mayprovide one or more of these proteins at a level that enhance orfacilitate such swelling.

The invention is described in more detail in the following examples,These examples show only a few concrete applications of the invention.It is self evident for one skilled in the art to create several similarapplications. Hence the examples should not be interpreted to narrow thescope of the invention only to clarify the use of the invention.

EXAMPLES Example 1

Shake Flask and Fermentor Cultivations

For maintenance, the strains ALKO4179, ALKO4124, ALKO4237, ALKO4265 andALKO4125 were streaked on sporulation agar (ATCC medium 5, American TypeCulture Collection, Catalogue of Filamentous Fungi, 18th edition, eds.,S. C. Jong and M. J. Edwards, (1991): 1 liter contains 1 g yeastextract, 1 g beef extract, 2 g tryptose, a trace amount of FeSO₄, 10 gglucose and 15 g agar; the pH was 7.2. Agar slants were incubated at 45°for 3-6 days.

For the applications tests of ALKO4237 Examples 3 and 4), a colony wasinoculated in 500 ml of the following mineral medium (Moloney, A. P. etal., Biotechnol. Bioeng. 25:1169 (1983)): 1 liter contains 15 g KH₂PO₄,15 g (NH₄)₂S₄, 2.4 ml of 1 M MgSO₄×7H₂0, 5.4 ml 1 M CaCl₂, 20 g Solkafloc, 15 g corn steep powder, 1 g yeast extract and 10 ml 100× traceelement solution 1, where 1 liter of 100× trace element solution 1contains 0.5 g FeSO₄×7H₂O, 0.156 g MnSO₄×H₂O, 0.14 g ZnSO₄33 7H₂O and0.49 g CoSO₄×7H₂O; the pH was adjusted to pH 6.5. Cultivation wasperformed at 45° C. for 3 days in a rotatory shaker (250 μm).Endoglucanase activity of about 20-25 nkat/ml was obtained.

Cellulase activity was routinely measured as endoglucanase activityaccording to Bailey, M. J. et al., Enzyme Microb. Technol. 3:153(1981)), using 1% (w/v) hydroxyethylcellulose, HEC (Fluka AG #54290) asa substrate. The assay conditions were, if not otherwise stated, pH 7.0and 50° C. with a 10 minute reaction time. One endoglucanase unit (1nkat=1 ECU) is defined as the amount of enzyme that produces reducingcarbohydrates having a reducing power corresponding to one nanomole ofglucose in one second from HEC under the assay conditions. However, withthe purified enzymes described in Examples 9-12, the assay conditions ofBailey et al., Enzyme Aicrob. Technol. 3:153 (1981) exceed the linearrange, and the assay was therefore modified as described in Example 10.With every strain, the filter paper activity assay (which measures thetotal hydrolysis of cellulose and indicates the presence ofcellobiohydrolase activity) was either under the reliable detectionlimit or very low.

For the determination of pH and temperature dependency (Example 2), aswell as for the application tests of the strains ALKO4179, ALKO4124,ALKO4265 and ALKO4125 (Examples 3 and 4), colonies were inoculated in500 ml of the modified thermomedium B (G. Szakacs, Technical Universityof Budapest, Hungary): 1 liter contained 6 g Solka floc, 6 g distiller'sspent wheat gain, 3 g oat spelt xylan, 2 g CaCO₂, 1.5 g soybean meal,1.5 g (NH₄)₂HPO₄, 1 g barley bran, 0.5 g KH₂PO₄.0.5 g MgSO₄×7H₂O, 0.5 gNaCl, 0.5 ml trace element solution 1 (1 liter contains: 1.6 g MnSO₂,3.45 g ZnSO₄×7H₂O, and 2.0 g CoCl₄×6H₂O) and 0.5 ml trace elementsolution 2 (I liter contains: 5.0 g FeSO₄×7H₂O and two drops ofconcentrated H₂SO₄); the pH was adjusted to pH 6.5. Cultivations wereperformed at 45° C. for 3 days in a rotatory shaker (250 rpm). Becausein thermomedium B the endoglucanase activities of the strains ALKO4179,ALKO4124, and ALKO4237 were about 5 nkat/ml, culture filtrates wereconcentrated about 10 fold in an Amicon concentrator using a cut-off-of30 kDa. Endoglucanase activity obtained with ALKO 4265 was about 20nkat/ml and with ALKO 4125 30-40 nkat/ml.

The 1 liter fermentor cultivation of ALKO4179 was performed in thefollowing medium: 1 liter contained 10 g Solka floc, 3 g cellobiose, 4 gcorn steep powder, 1.5 g (NH₄)₂HPO₄, 0.3 g MgSO₄×7H₂, 0.5 g NaCl, 2 gCaCO₃, 0.5 ml trace element solution 1 and 0.5 ml trace element solution2, 0.5 g KNO₃, 0.3 g CaCl₂, 1 g Tween 80; the pH was adjusted to pH 6.5.

The 1 liter fermentor cultivation of ALKO4124 was performed in themodified thermomedium B: 1 liter contained: 10 g Solka floc, 1 g Roth'sxylan, 40 g whey, 30 g soybean meal, 2 g CaCO₃, 5 g (NH₄)₂SO₄, 0.5 gKH₂PO₄, 1.0 g MgSO₄×7H₂O, 1.0 g NaCl, 1 g antifoam, 0.5 ml trace elementsolution 1 and 0.5 ml trace element solution 2.

The 1 liter fermentor cultivation of ALKO4237 was performed in themineral medium mentioned above. 10% (v/v) inoculum was used. pH wasmaintained at pH 6.5±0.4 by the addition of ammonia [12.5% (v/v)] andphosphoric acid [17% (v/v)]. The fermentation temperature was 45° C. Thefermentor (Biostat M, B. Braun, Melsungen, Germany) was stirred at 400rpm and the air flow as 1 vvm. The endoglucanase activities obtainedwere the following: ALKO4179 about 40 nkat/ml, ALKO4124 about 90 nkat/mland ALKO4237 about 30 nkat/ml. ALKO4265 and ALKO4125 were not cultivatedin a fermentor.

ALKO4179, ALKO4124, ALKO4237 and ALKO4125 were cultivated in a 100 literpilot fermentor in media and conditions described above. Endoglucanaseactivities obtained were about 40 nkat/ml with ALKO4179 and ALKO4237,about 90 nkat/ml with ALKO4124 and about 100 nkat/ml with ALKO4125.Culture filtrates were concentrated 10-20 fold in a Millipore PUF100ultra filter and a Pellicon Us cassette concentrator using a cut-off of10 kDa.

Example 2

Determination of the pH and the Temperature Dependence of theEndoglucanase Activities in the Culture Filtrates

For the determination of pH and temperature dependence, the strainsALKO4179, ALKO4124, ALKO4237, ALKO4265 and ALKO4125 were grown in themodified thermomedium B. Samples from the shake flask cultivations(culture filtrates) were diluted in 50 mM Mcllvain's buffers (50 mMcitric acid-100 mM Na₂HPO₄) of pH range 4.5-8.5. The final pH values ofthe culture filtrate buffer mixtures were 4.3, 5.4, 6.3, 7.3, 8.1 and8.7 for the strain ALKO4179; 4.3, 5.4, 6.4, 7.3, 8.1 and 8.5 for thestrain ALKO4124; 4.4, 5.3, 6.2, 7.1, 8.0 and 8.5 for the strainALKO4237; 4.3, 5.4, 6.3, 7.2, 8.1 and 8.5 for the strain ALKO4265 and4.3, 5.4, 6.4, 7.3, 8.1 and 8.5 for the strain ALKO4125. BSA was addedas a protein carrier to the concentration of 100 μg/ml. Pepstatin A andphenyl methyl sulphonyl fluoride (PMSF) were added as proteaseinhibitors at 10 μg/ml and 174 μg/ml, respectively. Endoglucanaseactivity was measured at each pH at 50° C. with 60 minutes reactiontime. The endoglucanase activity of ALKO4179 exhibited more than 90% ofits maximum in the pH range of about 4.5-7.5, the maximum activity wasdetected at about pH 5.4-6.3 (FIG. 1A). The endoglucanase activity ofALKO4124 exhibited more than 80% of its maximum activity in the pH rangeabout 5.5-7.5, the maximum activity was detected at about pH 6.4 (FIG.2A). The endoglucanase activity of ALKO4265 exhibited more than 80% ofits maximum activity in the pH range about 4.5-7.0, the maximum activitywas detected at about pH 5.5-6.5 (FIG. 4A). The endoglucanase activityof ALKO4237 exhibited more than 80% of its maximum in the pH range ofabout 4.5-6.0, the maximum activity was detected at about pH 5.3 (FIG.3A). The endoglucanase activity of ALKO4125 exhibited about 90% of itsmaximum in the pH range of about 4.5-7.5, the maximum activity wasdetected at about pH 6.5 (FIG. 5A).

For the temperature dependency determination of the endoglucanaseactivity, samples from the culture filtrates were diluted in 50 mMMcllvain's buffer, pH 7.0. BSA was added as a protein carrier to theconcentration of 100 μg/ml. Pepstatin A and phenyl methyl sulphonylfluoride (PMSF) were added as protease inhibitors to 10 μg/ml and 174μg/ml, respectively. The final pH values of the culture filtrate buffermixtures were 7.3 (ALKO4179, ALKO4124 and ALKO4125) and 7.2 (ALKO4237and ALKO4265). Samples were incubated at 40° C., 50° C. and 60° C. for60 minutes. The maximum endoglucanase activity of ALKO4179 was detectedat 50° C. and 60° C., about 30% of the activity was retained at 40° C.(FIG. 1B). The maximum endoglucanase activity of ALKO4124 was detectedat 60° C., about 70% of the activity was retained at 50° C. and 30% at40° C. (FIG. 2B). The maximum endoglucanase activity of ALKO4237 wasdetected at 60° C., about 60% of the activity was retained at 50° C. and40% at 40° C. (FIG. 3B). The maximum endoglucanase activity of ALKO4265was detected at 60° C., about 50% of the activity was retained at 50° C.and 30% at 40° C. (FIG. 4B). The maximum endoglucanase activity ofALKO4125 was detected at 60° C., about 80% of the activity was retainedat 50° C. and 70% at 40° C. (FIG. 5B).

Example 3

Indigo Dye Release in Neutral Conditions

Cellulase preparations derived from the strains ALKO4179, ALKO4124,ALKO4237, ALKO4265 and ALKO4125 (Examples 1 and 2) were tested for theirability to release dye in neutral conditions from the indigo dyedcotton-containing denim fabric to give a stone-washed look. Commercialacid cellulase product Ecostone L (Primalco Ltd, Biotec, Finland) wasused as a control.

Denim fabric was obtained from Lauffenmuehl (Germany). Test fabric wasprewashed 10 min at 60° C. with Ecostone A 200 (1 m/liter, Primalco Ltd,Biotec, Finland). The fabric was then cut into 12×12 cm swatches. Thecolour from both sides of the fabric swatches was measured asreflectance values with the Minolta (Osaka, Japan) Chroma Meter CM 1000RL*a*b* system.

Cellulase treatments were performed in LP-2 Launder-Ometer (Atlas,Illinois, USA) as follows. About 7 g of denim swatches were loaded intothe 1.2 liter container that contained 200 ml of 0.05 Mcitrate/phosphate buffer at pH 7, or, 0.05 M citrate buffer at pH 5.2.0.06 ml of 10% Berol 08 (Berol Nobel AS, Sweden) was added as asurfactant.

A quantity of steel balls were added into each container to help thefiber removal. Finally the cellulase solutions were added to thecontainer as endoglucanase activity units (Example 1). The containerswere then closed and loaded into a 50° C. Launder-Ometer bath. TheLaunder-Ometer was run at 42 rpm for 2 hours.

After removing swatches from the containers they were soaked for 10 minin 200 ml of 0.01 NaOH and rinsed for 10 min with cold water. Swatcheswere then dried for 1 hour at 105° C. and air dried overnight The colorfrom both sides of the swatches was measured with the Minolta ChromaMeter. Results from the color measurements of treated denim fabrics areshown in Table 1.

TABLE I Color Measurement of Denim Fabrics Treated with DifferentCellulase Preparations Right side Reverse side Source of ECU/g of theFabric of the Fabric Enzyme of fabric L b delta E L b delta E pH 7* — —2.3 0.8 3.1 1.5 0.1 0.9 ALKO4237 200 6.4 3.3 7.6 2.4 1.7 3.2 400 7.7 3.88.1 2.5 1.8 3.0 ALKO4179 200 5.5 2.4 6.4 2.8 1.9 3.0 400 4.6 2.8 5.1 2.21.5 3.0 ALKO4124 200 4.8 2.8 6.1 3.3 1.2 2.5 400 ND ND ND ND ND NDALKO4125 200 4.0 2.7 5.6 2.3 1.5 2.3 400 ND ND ND ND ND ND ALKO4265 2002.2 3.6 5.1 −4.9  6.6 9.2 400 ND ND ND ND ND ND Ecostone L 200 1.6 0.71.6 0   1.7 1.6 400 1.6 0.9 1.8 −1.9  2.2 2.8 pH 5.2** Ecostone L 200 2.01  2.33  3.30 −2.74  4.35  4.71 400  3.19  2.76  4.35 −2.56  4.83 6.71 L: Lightness unit of the fabric after the treatment minuslightness unit of the fabric before the treatment. b: Blueness unit ofthe fabric after the treatment minus blueness unit of the fabric beforethe treatment. delta E: Color difference in the L*a*b* color spacebetween the specimen color and the target color (target fabric =untreated denim fabric). ND = not done. *the ECU activity was measuredat pH 7.0. **the ECU activity was measured at pH 4.8.

To compare the final look of the denim fabrics after washing withdifferent cellulase preparations, the color from both sides (reverseside and right side) of the fabrics was measured. From the results shownin Table I, it can be seen that the lightness and blueness units areclearly increased on the right side of the garments washed withpreparations of ALKO4179, ALKO4124, ALKO4237 and ALKO4125 cellulases,showing a good stone-washed effect. The blueness unit was also increasedon the right side of the fabric washed with the ALKO4265 preparation butthere was no increase in the lightness unit. This is probably becausethe enzyme does work at this pH but at the same time causes a lot ofbackstaining. There was no stone washing effect on the fabric withcommercial acid product Ecostone L at pH 7 at this ECU activity.

In this study, backstaining on the reverse side of the fabric is used asan indication of the degree of backstaining on the right side of thefabric. To quantify the level of backstaining, the color was measured onthe reverse side of the fabric before and after the cellulase treatment.As shown in Table I, when the ECU amounts are the same, there waspractically no backstaining in the fabrics treated with the ALKO4179,ALKO4124, ALKO4237 and ALKO4125 preparations when compared to thefabrics treated with ALKO4265 or Ecostone L (pH 5.2 and 7) preparations.

Example 4

Dye Release in Neutral Conditions, No Berol

The experimental set-up was as described in Example 3 except that noBerol was used. Results from the color measurements of treated denimfabrics are shown in Table II.

TABLE II Color Measurement of Denim Fabrics Treated with DifferentCellulase Preparations — no Berol Right side Reverse side Source ofECU/g of the Fabric of the Fabric Enzyme of fabric L b delta E L b deltaE pH 7* — — 2.1 0.5 2.2 1.7 −1.1  2.0 ALKO4237 200 5.5 3.1 7.0 1.8 2.33.5 ALKO4179 200 4.4 3.2 5.6 1.4 2.2 2.7 ALKO4124 200 4.2 2.9 5.0 1.12.0 2.4 ALKO4125 200 3.5 2.6 4.4 1.6 1.4 2.5 ALKO4265 200 3.3 3.3 5.3−5.7  6.6 10.0  Ecostone L 200 1.4 0.9 1.7 0.3 1.4 1.8 400 1.4 0.8 1.7−0.1  1.7 1.8 pH 5.2** Ecostone L 200 2.0 2.1 2.9 −4.0  4.8 5.4 L:Lightness unit of the fabric after the treatment minus lightness unit ofthe fabric before the treatment. b: Blueness unit of the fabric afterthe treatment minus blueness unit of the fabric before the treatment.delta E: Color difference in the L*a*b* color space between the specimencolor and the target color (target fabric = untreated denim fabric). ND= not done. *the ECU activity was measured at pH 7.0. **the ECU activitywas measured at pH 4.8.

When compared with results obtained with the inclusion of Berol (Example3), the data in Table II show that almost the same stone-washing effectcan be achieved with the ALKO4179, ALKO4124, ALKO4237 and ALKO4125cellulase preparations in the absence of the helping agent Berol.

Example 5

Backstaining in Denim Wash with Different Cellulases

In the literature, it is reported that backstaining is dependent on pHand/or the type of enzyme. However, as shown herein, it was found thatbackstaining depends only indirectly on pH (FIGS. 6A and 6B and 7A and7B).

Two neutral cellulase preparations from ALKO4237 and from ALKO4265 andacid cellulase product Ecostone L were studied in small scale denim washwith an equal enzyme dosage at pH 5 and pH 7. The stonewash effect wasdetermined by measuring the increase of lightness and blueness asreflectance units on the right side of the fabric and backstaining(redeposition of indigo on the surface of fibers) was determined asblueness increase and lightness decrease on the reverse side. At pH 7,the neutral cellulases from ALKO4237 caused a clear increase inlightness and blueness on the right side and no backstaining wasobserved (FIG. 6A and 6B). A similar stonewash effect was found at pH 5but with a slight backstaining. At pH 7, the other neutral cellulase,ALKO4265, brightened blueness on the right side but backstainedintensively on the reverse side. At pH 5 similar effects were obtainedwith both ALKO4265 and ALKO4237 preparations. At pH 7, the acidcellulase did not backstain or impart a lightness on the right side(when using similar endoglucanase activity dosages as with ALKO4265 andALKO4237, FIG. 7A and 7B, 1× dosage), probably because it did not workat this pH. On the other hand, at pH 5, lightness and blueness wereincreased on the right side and backstaining was clearly perceptible onthe reverse side. Based on these results, backstaining can occur at bothpH values depending on the cellulase preparation used.

EXAMPLE 6

Use of the Neutral Cellulase-Containing Enzyme Preparations inBiofinishing of Colton-Containing Woven Fabric

100 % cotton woven fabric was subjected to treatment with ALKO4237(Example 1) and ALKO4467 cellulases in Launder-Ometer. ALKO4467 is aUV-mutant with higher cellulase activity derived from ALKO4125.

100 % cotton woven fabric (obtained from Pirkanmaan Uusi Värjäämö Ltd)was pretreated as in Example 7. The cellulase treatment conditions wereas described in Example 3 except that no Berol was used and the liquidratio was 1:15 (volume of liquid per weight of fabric). Cellulases weredosed as ECU activity units (Example 1).

The following methods were used for evaluation of the effect of theenzyme preparations in biofinishing of cotton fabric: Weight loss of thetreated fabrics was defined as percentage from weight of the fabricbefore and after the test (before weighing the fabrics were conditioningin a atmosphere of 21+2° C. and 50+5% RH). Evaluation of the surfacecleaning effect of the enzyme treated fabrics was performed by a panelconsisting of three persons. The fabrics were ranked on a score from 1to 5, where 5 gave a clean surface. The Martindale Rubbing method(SFS-4328) was used for evaluation of pilling. Pilling was evaluated bya panel after 200 and 2000 cycles of abrasion (1=many pills, 5=nopills).

In Table III is shown that treatment of the cotton fabric with ALKO4237and ALKO4467 cellulase preparations results in a good surface cleaningand marked reduction in the pilling tendency at both pH 5 and 7.

TABLE III Weight loss, surface cleaning effect and pilling tendency ofthe cotton fabrics treated with neutral cellulases in Launder-Ometerweight surface pilling dosage time loss cleaning 200 2000 preparationECU/g h pH % effect cycles cycles — — 1 5 0 1.0 1.0 1.0 ALKO4237 200 1 52.3 3.5 4.0 3.8 ALKO4237 400 1 5 3.2 3.5 4.0 3.8 ALKO4467 200 1 5 1.22.5 3.7 3.4 ALKO4467 400 1 5 1.9 2.8 3.7 3.4 — — 2 5 0.1 1.0 1.0 1.0ALKO4237 200 2 5 4.4 4.0 4.2 4.1 ALKO4237 400 2 5 6.0 4.3 4.2 4.3ALKO4467 200 2 5 3.0 3.5 4.0 3.8 ALKO4467 400 2 5 4.0 3.8 4.0 3.9 — — 17 0 1.0 1.0 1.0 ALKO4237 200 1 7 2.5 3.0 3.7 3.5 ALKO4237 400 1 7 3.84.0 4.0 3.9 ALKO4467 200 1 7 0.8 2.0 3.5 3.3 ALKO4467 400 1 7 1.4 2.03.6 3.7 — — 2 7 0.1 1.0 1.2 1.1 ALKO4237 200 2 7 4.8 4.0 3.8 4.0ALKO4237 400 2 7 6.0 4.3 4.0 4.3 ALKO4467 200 2 7 2.2 2.5 4.0 3.4ALKO4467 400 2 7 3.0 3.3 3.8 3.7

Example 7

Use of the Neutral Cellulase-Containing Enzyme Preparations of theInvention in Biofinishing

7a. Use of Enzyme Preparations in the Biofinishing of Woven Fabric andKnit

100% cotton woven fabric or 100% cotton knit are subjected to treatmentwith the cellulases of the invention (Example 1) in a semi-industrialdrum washer (Esteri 20 HS-P). The treatment conditions are as follows:

A. Pretreatment (only for woven fabrics)

60° C., 10 minutes, Ecostone A200 (Primalco Ltd, Biotec, Finland) 1 ml/lwater.

B. Enzyme treatment

temperature 50-60° C., pH 7;

liquid ratio 5-20:1 (volume of liquid per weight of fabric);

treatment time 20-90 minutes, preferably 30-60 minutes; and

enzyme dosage 50-900 nkat/g fabric or knit, preferably 200-600 nkat/gfabric or knit.

C. “After-washing” treatment

40° C., 10 minutes, alkaline detergent

D. Drying treatment

The following standard methods are used for evaluation of the surfacecleaning effect of enzyme preparations: The Martindale Rubbing Method(SFS-4328) and the Laundering Durability Test (SFS-3378). Treatment withthe cellulase preparations of the invention results in a surfacecleaning effect, an improvement in the softness and smoothness of thefabric and knit and a reduction in the pilling tendency.

7b. Use of Enzyme Preparations in the Finishing of Lyocell Fabrics andKnits

The cellulase preparations of the invention can be used in fibrillationcontrol and different finishing processes of 100% lyocell fabrics andknits and blends thereof. The following treatment conditions insemi-industrial drum washer (Esteri 20 HS-P) are used in order to createthe peach effect on lyocell fabric:

A. Sodium carbonate 2.5 g/l; 60° C., treatment time of 60 minutes;

B. Rinse;

C. Enzyme treatment: temperature of 50-60° C., pH 7, liquid ratio5-20:1, treatment time 40-120 minutes, preferably 45-90 minutes, and anenzyme dosage of 100-1500 nkat/g fabric, preferably 400-800 nkat/gfabric;

D. Aftertreatment: Alkaline detergent wash at 40° C. for 10 minutes;

E. Rinse; and

F. Dry.

The result is a peach skin effect.

Example 8

Use of Enzyme Preparations in Biostoning

Denim garments were subjected to treatment with the neutral cellulasepreparations (Example 1) in a semi-industrial drum washer (Esteri 20HS-P) to give the garments a stonewashed appearance. About 1.0 kg ofdenim garments (contained two different kinds of fabric) were used permachine load.

The treatment conditions were as follows.

A. Desizing. 100 liters water, 60° C., 10 minutes; 100 ml Ecostone A200(Primalco Ltd, Biotec, Finland).

B. Cellulase Treatment 100 liter water, 50° C., 45 minutes; 10 g Berol08 (Berol Nobel AS, Sweden); 30 g citric acid +128 g Na₂HPO₄×2 H₂O togive pH 7.

Neutral cellulase preparations were dosed as endoglucanase activityunits (ECU, Example 1):

1. ALKO4237, 260 ECU/g of garment

2. ALKO4179, 260 ECU/g of garment

3. ALKO4124, 300 ECU/g of garment

4. ALKO4125, 250 ECU/g of garment

C. Afterwashing. Alkaline detergent wash, 40° C., 10 minutes.

D. Drying

The results were evaluated by visual appearance of the garments and bymeasuring the color as reflectance values with the Minolta Chroma MeterCM 1000R L*a*b system (Table IV). A good stonewashed effect was obtainedwith all these cellulase-treated garments. No backstaining (examined onthe inside of the garment) could be seen visually in any of thesecellulase-treated garments.

From the results of the color measurements shown in Table IV, it can beseen that the lightness and blueness units are clearly increased on theoutside of the garments washed with the neutral cellulase preparations,showing a good stonewashed effect.

TABLE IV Color Measurement of Denim Garments with Different CellulasePreparations Source of Outside of the garment Inside of the garmentEnzyme L b L b A. Fabric 1 untreated 24.1 −8.5 57.1 0.17 washed without21.4 −14.0 54.5 −4.3 cellulase ALKO4237 26.7 −17.3 56.5 −4.9 ALKO417926.8 −17.0 56.3 −4.5 ALKO4125 28.0 −17.4 57.8 −4.1 ALKO4124 26.4 −17.557.1 −4.8 B. Fabric 2 untreated 22.5 −8.3 57.6 0.66 ALKO4237 25.0 −16.356.1 −4.3 ALKO4179 25.0 −15.8 55.4 −4.4 ALKO4125 26.7 −17.0 56.8 −4.0ALKO4124 25.6 −17.0 56.4 −4.0 L = Lightness unit of garment after thetreatment (the higher the value, the lighter the garment). b = Bluenessunit of garment after the treatment (the more negative value, the moreblueing in the garment).

Example 9

Purification of Neutral Cellulases

Concentrated growth medium from ALKO4237 was fractionated at 7° C. onDEAE Sepharose CL6B with a linear gradient from zero to 0.5 M NaCl in 25mM Tris/HCl pH 7.2. Four peaks of endoglucanase activity at pH 4.8 werefound. Peak I, containing about 10% of the recovered ECU, eluted atabout 150 mM NaCl, Peak II (about 30% of ECU) at 230 mM NaCl, Peak III(about 20% of ECU) at 270 mM NaCl and Peak IV (about 40% of ECU) at 320mM NaCl. Table V shows the results when these peaks were tested fortheir utility in biostoning at neutral pH and 50° C.

These results show that on both an ECU basis and a total protein basis,Peak II was more effective than any other peak or than theunfractionated concentrate. A mixture of Peaks I and II containing 70ECU of each/g denim was also tested. This resulted in an L (right) valueof 7.3 and b (reverse) of 2.5. Thus, this mixture was more effectivethan either peak alone.

The purification procedure was scaled up to obtain homogenous samples ofsome of the desired proteins in these peaks. Concentrated ALKO4237growth medium (4.5 liters) was fractionated with ammonium sulphate. Theproteins that precipitated between 17 g and 42 g of ammonium sulphateper 100 ml of concentrate were suspended in 0.9 liter of 25 mM Tris/HClpH 7.2 containing 0.25 mM EDTA and then diluted with water to aconductivity of 4 mS/cm and adjusted with 1M NaOH to pH 8.0. Theresulting solution (about 45 liters) was pumped at 150 ml/min through a6.3 liter column of DEAE-Sepharose FF™ at room temperature. Peak Iendoglucanase activity did not bind under these conditions. Boundproteins were eluted at 110 ml/min with a linear gradient from 0.0 to0.5 M NaCl in 20 liters of 25 mM Tris/HCl pH 7.7 containing 0.25 mMEDTA. Peak II endoglucanase eluted at about 14 mS/cm. Instead of theseparate Peaks III and IV seen with small scale separations in DEAE inthe cold room, a single peak, called Peak III/IV, eluted at about 25mS/cm.

TABLE V Indigo Dye Release by DEAE-Sepharose ™ Pools in NeutralConditions ADDITION None Concentrate Peak I Peak II Peak III Peak IVECU/g 0 100 200 310 185 340 97 260 95 190 mg/g 0 10 20 41 9 26 24 46 510 L (right) 2.9 5.2 7.0 5.5 7.3 10.3 4.4 5.8 3.9 4.3 b (reverse) 0.42.6 3.5 3.5 2.9 2.5 0.9 1.4 0.1 0.5 The parameter L (right) indicatesthe lightening of the right side of the blue denim, and b (reverse)indicates the blueing of the reverse side (i.e., backstaining). Thefabric was washed in the LP-2 Launder-Ometer and then measured with theMinolta ChromaMeter, as described in Example 3, except that no Berol wasused and the buffer that was used was 0.05M McIlvaine pH 7 (see Data forBiochemical Research, Dawson, R., et al., eds., 1969, Oxford Univ.Press). The dosage is shown as # both ECU/g of denim and mg protein/g ofdenim.

Proteins in Peak II (3.5 liters) were precipitated with ammoniumsulphate (450 g/liter) and suspended in 170 ml 25 mM PIPES/KOH pH 6.0containing 1 mM EDTA Portions of this material were transferred to 25 mMsodium acetate pH 4.0 containing 1 mM EDTA by gel-filtration on a 5×29cm column of G25 Sephadex™ (coarse) and then fractionated onSP-Sepharose™. FIG. 8 shows the result that was obtained when 11.7 g ofthese proteins was applied to a 4.5×31 cm column of SP-Sepharose™ in 25mM sodium acetate pH 4.0 containing 1 mM EDTA at 150 ml/h and the columndeveloped at 75 ml/h with a linear gradient from 0.0 to 0.4 M NaCl in3.4 liters of the same buffer. Most of the endoglucanase eluted at 0.2 MNaCl. The modified assay described in Example 10 was used. When activefractions were stored at 7° C., a crystalline precipitate appeared inthem and contained nearly all the endoglucanase activity. Activefractions (15 ml) in which crystallization was slow, were induced toform crystals by seeding with 30 μl of suspension from fractions alreadycontaining crystals. After 2 to 3 days, the crystals were collected bycentrifugation, washed with 25 mM PIPES/KOH pH 6.0 containing 1 mM EDTAand dissolved in 25 mM Tris/HCl pH 7.2 containing 0.25 mM EDTA. Analysisby SDS-PAGE showed the washed crystals contained a virtually homogenousprotein with an apparent molecular mass close to 20 kDa (the error inSDS-PAGE estimations of molecular mass is at least ±10%, and may be muchgreater for unusual proteins). This protein is called the 20K-cellulase.Contaminating protein could also be removed by gel-filtration on G50Sephadex™ in 50 mM PIPES/KOH pH 6.0 containing 1 mM EDTA. An example ofthis is shown in FIG. 9, where unwashed crystals were purified bygel-filtration The endoglucanase activity co-eluted with the 20 kDaprotein well after the cytochrome c (11.2 kDa) volume, showing that this20 kDa protein is abnormally retarded by interaction with Sephadex™.

Proteins in Peak III/IV were precipitated with ammonium sulphate andtransferred to 25 mM sodium acetate pH 4.0 containing 1 mM EDTA in thesame way as described for the Peak II proteins. Upon transfer to 25 mMsodium acetate pH 4.0, a large precipitate formed and was discarded. Theactive supernatant was fractionated on SP-Sepharose™. At low proteinloading (e.g. 200 mg protein to a 2.5×11 cm column as shown in FIG. 10,most of the endoglucanase activity bound to the column and was elutedwith a NaCl gradient at about 50 mM NaCl. This active peak was followedby a second peak of inactive protein.

SDS-PAGE analysis showed that the active and inactive peaks bothcontained several proteins, including proteins with apparent molecularmasses close to 50 kDa that could not be distinguished from each otherby SDS-PAGE. Both peaks were further purified by chromatography onPhenyl Sepharose™.

The active fractions (fractions 15 to 18 in FIG. 10) were pooled,adjusted to 50 mM PIPES/KOH pH 6.0 (by addition of 0.25 M PIPES/KOH pH6.0) and 15 g % ammonium sulphate (by addition of solid ammoniumsulphate) and applied to a 1.5×8.5 cm column of Phenyl Sepharose™equilibrated with 25 mM PIPES/KOH pH 6.0 containing 1 mM EDTA and 15 g %of ammonium sulphate. The column was developed with a linear gradientfrom 15 to 0 g % ammonium sulphate in 104 ml of 25 mM PIPES/KOH pH 6.0.After the end of the gradient, the column was further washed with 25 mMPIPES/KOH pH 6.0. Two protein peaks eluted on the gradient, first asmall peak of inactive protein and then a major peak containing most ofthe endoglucanase activity. SDS-PAGE analysis (FIGS. 11A and B) showedthat both peaks contained essentially homogenous proteins with apparentmolecular masses close to 50 kDa (i.e., they migrate slightly slowerthan the BioRad prestained ovalbumin standard, which has an apparentmolecular mass of 47 kDa). These two proteins could not be distinguishedby the inventors' SDS-PAGE analyses, even when they were run together asmixtures. The protein in the active peak was called 50K-cellulase andthe protein in the inactive peak was called 50K-protein B. Largeramounts of 50K-cellulase B were obtained by fractionation of the second(and inactive) peak eluted from SP-Sepharose™ (fractions 19 to 23 inFIG. 10) on Phenyl Sepharose™ in exactly the same way as described abovefor the active fractions.

Production of still larger amounts of 50K-cellulase and 50K-cellulase Bwas facilitated by overloading the SP-Sepharose™ column. For example,when 15 g of protein was applied to a 4.5×31 cm column of SP-Sepharose™,instead of binding to the column, the 50K-cellulase was apparentlydisplaced by more strongly bound proteins, and eluted before the NaClgradient. This material was already highly purified, and homogenous50K-cellulase was isolated from it by chromatography on PhenylSepharose™ as described above.

In order to speed up the purification of larger amounts of 50K-cellulasethe SP-Sepharose and Phenyl Sepharose columns were reversed. Afteradjusting the ammonium sulphate concentration to about 15 g%, theproteins precipitated in Peak III/IV were applied into Phenyl Sepharoseas described before. With high overloading (e.g. 17 g of protein appliedto a 3.2×25 cm column of Phenyl Sepharose) most of the total protein ranthrough the column, but 50K-cellulase (containing most of theendoglucanase activity) was bound and eluted at the end of lineargradient from 15 to 0 g % of ammonium sulphate in 25 mM PIPES/KOH pH6.0. Western analysis with a rabbit antiserum recognizing 50K-cellulaseB showed that the 50K-cellulase B eluted just before 50K-cellulase.Further purification was achieved by fractionation on SP-Sepharose asdescribed earlier. In this reversed order of SP-Sepharose and PhenylSepharose the proteins in Peak III/IV precipitated with ammoniumsulphate could be applied directly to the next purification step withoutremoving salt. The large protein precipitate, which appeared upontransfer of the concentrated proteins in Peak III/IV directly into 25 mMsodium acetate pH 4.0 for SP-Sepharose, could also be avoided this way.As the 50K-cellulase only just binds to SP-Sepharose, the preceedingfractionation on Phenyl Sepharose markedly reduced the apparentlyinterfering total protein load on SP-Sepharose.

50K-cellulase and 50Kellulase B were each tested in the Launder-Ometerto see if they are responsible for the beneficial effects of Peak IVreported in Example 10. Both proteins were found to have beneficialeffects (Table VI). At the low concentrations used in this experiment,they did not themselves increase the release of indigo dye from theouter face of the denim (i.e., L_(right) did not increase) but theyeffectively decreased the back-staining of dye onto the inner face ofthe denim (L_(reverse) became more positive and b_(reverse) becamesmaller) especially when used together with 2K-cellulase.

The 20K-cellulase performed well in Launder-Ometer tests at pH 5 as wellas at pH 7. At pH 5, 0.2 mg of 20K-cellulase per g of denim increasedL_(right) from 3.2 to 5.2. Addition of 50K-cellulase at 0.1 mg per gramof denim together with the 20K-cellulase also decreased the backstainingat pH 5 (L_(reverse) and b_(reverse) changed from 0.0 and 2.6 with2K-cellulase alone to 1.3 and 1.5, respectively, with the mixture of20K-and 50K-cellulases).

TABLE VI Indigo Dye Release by 20K-cellulase, 50K-cellulase and50K-cellulase B Conditions were the same as in Table V. The dose isshown as mg protein per gram of denim. Dose Sample (mg/g) L_(right)L_(reverse) b_(reverse) Buffer alone — 2.8 −0.6 1.6 20K-cellulase 0.185.6 −1.0 4.0 0.09 4.8 −1.5 3.3 50K-cellulase 0.15 2.6 −0.3 1.0  0.0753.0 0.4 1.3 50K-cellulase B 0.31 2.8 1.3 0.8 0.15 2.7 1.5 0.520K-cellulase + 0.18 + 0.075 5.6 0.3 2.5 50K-cellulase 0.09 + 0.075 5.10.3 2.1 20K-cellulase + 0.18 + 0.15  4.7 0.0 3.0 50K-cellulase B

Example 10

Properties of the 20K-cellulase

Although polyclonal antibodies prepared against cellulases purified fromTrichoderma reesei (designated anti-EGI, anti-CBHI and anti-CBHIIantibodies) recognized proteins in the ALKO4237 growth medium, there wasonly a very weak cross-reaction with pure 20K-cellulase under the sameconditions of Western blot analysis.

When growth medium from ALKO4237 was probed on Western analysis withantiserum raised in rabbits against pure 20K cellulase, a strong band atabout 35 kDa was observed in addition to the 20 kDa band. No apparentendoglucanase activity could be detected for this 35 kDa protein. Also,a weaker band was seen immediately ahead of the 20 kDa band (FIG. 14).

ALKO4124 gave an almost identical pattern as ALKO4237, indicating thatthis and other fungi probably contain cellulases very similar to the20K-cellulase of the present invention.

Amino acid sequences of tryptic peptides derived from 20K-cellulases areshown in FIG. 17. #429 corresponds to SEQ ID NO: 1, #430 corresponds toSEQ ID NO: 2, #431 corresponds to SEQ ID NO: 3, #432 corresponds to SEQID NO: 4, #433 corresponds to SEQ ID NO: 5, #439 corresponds to SEQ IDNO: 15, fr 9 corresponds to SEQ ID NO: 7, fr 14 corresponds to SEQ IDNO: 8, fr 16 corresponds to SEQ ID NO: 9, fr 17 corresponds to SEQ IDNO: 10, fr 28 corresponds to SEQ ID NO: 11, and fr 30 corresponds to SEQID NO: 12.

Purified 20K-cellulase performed well in biostoning at neutral pHwithout the addition of other enzyme activities as shown in Table VII.

TABLE VII Biostoning by Purified 20K-cellulase Conditions were as in theexperiment shown in Table V. Dosage is shown as mg protein/g denimfabric. “Whole medium” indicates the unfractionated ALKO4237concentrated growth medium. Addition Dosage L_(right) b_(right)L_(reverse) b_(reverse) Buffer 0.0 3.6 0.1 0.5 0.6 20K 0.72 8.9 2.9 −1.14.7 20K 0.25 6.0 2.3 −0.5 3.6 20K 0.07 5.3 1.7 −0.4 2.9 Whole medium 206.1 2.8 −2.9 5.5

Compared to the unfractionated medium, 20K-cellulase resulted in thesame degree of lightening (L_(right)=6.0-6.1) at 1/80th the proteindosage. Further, there was less backstaining onto the reverse side faceof the fabric (L_(reverse)=−0.5 compared to −2.9 and b_(reverse)=3.6compared to 5.5). Fabric treated with 20K-cellulase had an agreeablesoft texture.

Although 20K-cellulase performed surprisingly well without otheradditions, even better fabric appearance and texture resulted when 20Kwas used together with the DEAE-Sepharose pools I, III or IV (TableVIII).

TABLE VIII Synergy in Biostoning Between 20K-cellulase and EndoglucanasePools Eluted from DEAE-Sepharose Conditions were as in Table V. AdditionDosage L_(right) b_(right) L_(reverse) b_(reverse) Buffer 0.0 3.8 0.2−0.7 1.5 20K 0.18 5.8 2.3 −2.2 5.5 Pool I 15 5.1 1.9 −3.1 5.7 Pool III47 5.2 1.6 −0.1 2.6 PooI IV 14 5.6 0.9 0.4 1.8 20K + Pool I 15.18 7.12.8 0.7 3.3 20K + Pool III 47.18 7.6 3.1 −1.7 5.3 20K + Pool IV 14.188.6 2.6 0.8 3.2 Whole medium 20 5.7 2.4 −4.1 5.9

The mixtures of 20K-cellulase with Pools I, III and IV caused morelightening (increased L_(right)) than either component alone. At leastfor the combination of 20K-cellulase with Pool IV, it is clear that thisis because of synergy and not merely an additive effect. Further, thebackstaining with all mixtures was actually less (L_(reverse) morepositive, b_(reverse) less) than the backstaining observed with20K-cellulase alone. The combination of 20K with Pool IV wasparticularly effective. Pool IV contains many proteins, one of which (a50 kDa polypeptide) copurifies with endoglucanase activity duringchromatography of Pool IV on Sephadex G100 and S-Sepharose. While goodbiostoning is achieved with 20K-cellulase alone, better results arepossible with 20K-cellulase plus one or more proteins purified from PoolIV. Biostoning with mixtures of the 20K-cellulase and the 50K-cellulaseand the 50Kellulase B purified from Pool III/IV have already beenpresented (Table VI in Example 9). Therefore, the present invention isnot limited to the use of only the 20K-cellulase. Other proteins in theALKO4237 medium are useful alone or in suitable combinations.

In the standard endoglucanase assay described by Bailey et al. (1981,loc. cit.), the enzyme amount is chosen that produces, in 10 min and pH4.8 (0.05 M Na-citrate buffer), about 0.6 mM reducing equivalents from1% hydroxyethylcellulose, resulting in a final absorbance change (ΔA₅₄₀)of between 0.2 and 0.25. This far exceeds the range in which ΔA₅₄₀ isproportional to the amount of 20K-cellulase.

Therefore, the procedure was modified as follows. Enough enzyme was usedto produce about 0.2 mM reducing equivalents in 10 min in 0.05 M HEPESbuffer (pH 7.0). To reach the threshold concentration of reducingequivalents above which color is formed in the DNS system, 0.12 mMglucose was freshly added to the stock DNS reagent. This method (calledthe “modified” method) was used when characterizing the endoglucanaseactivity of the 20K-cellulase and also the 50K-cellulase. With 1 %hydroxyethylcellulose as substrate, the range in which ΔA₅₄₀ isproportional to the amount of 20- and 50K-cellulase is relativelynarrow, and so 2% carboxymethylcellulose was taken as an alternativesubstrate. With 2% carboxymethylcellulose, the range of linearcorrelation between ΔA₅₄₀ and the amount of 20K- and 50K-cellulase wasbroader than with 1% hydroxyethylcellulose. The endoglucanase activitydetermined with 2% carboxymethylcellulose was about 8-10-fold for20K-cellulase and about 50-fold for 50K-cellulase compared with thatdetermined with 1% hydroxyethylcellulose.

No activity of 20K-cellulase was detectable for4-methylumbelliferyl-β-D-lactoside, a characteristic substrate ofcellobiohydrolases. The activity towards filter paper was also very low,but detectable.

The 20K-cellulase was relatively heat stable. It was incubated at 7μg/ml and 100° C. in 25 mM Tris-HCl, 0.2 mM EDTA, for 30 or 60 min. andthen assayed at pH 7.0 and 50° C. 52% and 35% respectively, of theendoglucanase activity remained at pH 7.2. 40% and 22%, respectively,remained at pH 8.8. (These pH values were measured at room temperature;the actual pH at 100° C. is somewhat lower.) At 80° C., pH 7.2, 70% ofthe activity remained for 60 min.

These results indicate that the enzyme is suitable for applications inwhich it may be (e.g., accidentally) exposed to elevated temperatures.As well as being resistant to irreversible inactivation at hightemperatures, the enzyme exhibited an optimum temperature of 70° C.during 10 min. assays at pH 7.0 (FIG. 15). The decreased activityobserved above 70° C. was mainly due to a reversible change in enzymeconformation: the enzyme recovered most of its activity when returned to50° C.

At 50° C., the 20K-cellulase exhibited 80% or more of its maximumactivity throughout the pH range 4 to 9, and nearly 50% at pH 10. Thiswas the case in both 10 min. (FIG. 16A) and 60 min. (FIG. 16B) assays.These figures also show the pH dependence of the enzyme at 70° C. With10 min. assays, the enzyme was more active at 70° C. than it was at 50°C. over the range pH 4.5 to 8 and about equally active at pH 10 (FIG.16A). With 60 min. assays (i.e., approaching commercial conditions), theenzyme was more active at 70° C. than it was at 50° C. between pH 5.5and 7.5. However, it was only slightly less active at 70° C. than at 50°C. up to pH 10. In practice, this means that the enzyme can be usedequally well over a wide range of pH and at temperatures up to at least70° C.

Example 11

Properties of the 50K-cellulase

Pure 50K-cellulase had both endoglucanase activity (againsthydroxyethylcellulose) and cellobiohydrolase activity (against4-methylumbelliferyl-β-D-lactoside, assayed essentially as described byvan Tilbeurgh et al, in Methods in Enzymology [1988] vol. 160, pp45-59). A sample of the pure enzyme with an A₂₈₀ of 1.8 contained 2030ECU/ml and 300 PCU/ml at pH 7.0 and 50° C. (one PCU is the amount ofactivity that liberates 1 nmol of methylumbelliferone per second).

In Western analyses, 50K-cellulase was strongly recognized by antiserum(KH 1057) raised against endoglucanase I of T. reesei, but only weaklyby antisera (KH 1050 and KH 1053, respectively) againstcellobiohydrolases 1 and II of T. reesei. It was not recognized by theantiserum raised against 20K-cellulase (FIG. 14). When the growth mediumof ALKO 4237 was probed in Western analyses with rabbit antiserum raisedagainst 50K-cellulase itself, only one obvious band (which had amolecular mass between 33 and 47 kDa) was seen in addition to the verystrong band at about 50 kDa.

The apparent molecular mass of 50K-cellulase by SDS-PAGE decreased byabout 2 to 5 kDa when the protein was treated with endoglycosidase Hf,indicating that the enzyme contains carbohydrate removable by thisendoglycosidase.

50K-cellulase was unusually resistant to tryptic digestion, indicatingthat it has an unusually stable structure. However, it was cleaved bytreatment with cyanogenbromide, and the resulting fragments could thenbe digested with trypsin or with lysylendopeptidase C. Sequences of someof the peptides so obtained are shown in Table IX.

TABLE IX Sequences of peptides isolated from the 50K- cellulase(uncertain residues in lower case) #507 (SEQ ID NO:13) VYLLDETEHR #509(SEQ ID NO:14) XXLNPGGAYYGT #563 (SEQ ID NO:15) MsEGAECEYDGVCDKDG #565(SEQ ID NO:16) NPYRVXITDYYGNS #603 (SEQ ID NO:17)DPTGARSELNPGGAYYGTGYXDAQ #605 (SEQ ID NO:18) XXVPDYhQHGVda #610 (SEQ IDNO:19) NEMDIXEANSRA #611 (SEQ ID NO:20) LPXGMNSALYLSEMDPTGARSELNP #612(SEQ ID NO:21) VEPSPEVTYSNLRXGEIXgXF #619 (SEQ ID NO:22)DGCGWNPYRVvITtDYYnN #620 (SEQ ID NO:23) LPCGMXSALY #621 (SEQ ID NO:24)ADGCQPRTNYIVLDdLlHPXXQ

The 50K-cellulase is a stable enzyme that exhibits endoglucanaseactivity over a wide range of pH values and at high temperatures, so itis suitable for use in many industrial conditions. At pH 7.0 and with 60min reaction times, it has an optimum temperature between 65 and 70° C.,and even with this long reaction time still exhibits, at 75° C., 50% ofthe activity observed at 50° C. (FIG. 12).

With 60 min reaction times, the pH optimum was very broad at 50° C.,with essentially constant activity between pH 4.4 and 7.0, andactivities at pH 9 and 10 equal to 50% and 30%, respectively, of that atpH 7.0. At 70° C., there was a clear optimum at pH 6, and, between pH 5and 7, the activity (with 60 min reaction times) was 3-fold or moregreater than that at 50° C. However, at pH 4.4 and pH values above 8,the activity was greater at 50° C. than at 70° C. (in 60 min assays),suggesting that the stability of the enzyme decreases at 70° C. rightside the pH range 5 to 7.5. The pH-dependence is illustrated in FIG. 13.

Example 12

Properties of 50K-cellulase B

No detectable endoglucanase activity could be measured for the50K-cellulase B (previously called 50K-protein B) withhydroxyethylcellulose or carboxymethylcellulose. At acidic pH, the50K-cellulase B had a low cellobiohydrolase activity, which (measuredwith 4-methylumbelliferyl-β-D-lactoside) at pH 5 was less than 0.1% ofthat of the 50K cellulase. In addition, the 50K-cellulase B had adetectable activity towards filter paper at pH 4.8 and acid swollen,amorphic Solca Floc-cellulose at pH 5 and 7 used in enzyme activitydeterminations.

In Western analyses, 50K-cellulase B was strongly recognized byantiserum (KH1050) raised against cellobiohydrolase I of T. reesei, butonly weakly by antisera against cellobiohydrolase II or endoglucanase Iof T. reesei or against the 50K-cellulase. It was not recognized byantiserum raised against the 20K-cellulase (FIG. 14). Table X showssequences of peptides isolated from 50K-cellulase B.

TABLE X Sequences of peptides isolated from the 50K- cellulase B(uncertain residues in lower case) #534 (SEQ ID NO:25) vGNPDFYGK #535(SEQ ID NO:26) FGPIGSTY #631 (SEQ ID NO:27) LSQYFIQDGeRK #632 (SEQ IDNO:28) FTVVSRFEENK #636 (SEQ ID NO:29) HEYGTNVGSRFYLMNGPDK

Example 13

Stability of Neutral Cellulases in Different Detergents

Stability of the neutral cellulase preparations were tested in threedifferent detergent solutions. The detergent solutions were OMO® Total(or OMO® Neste, Lever UK), OMO® Color (Lever S.A.) and Colour DetergentLiquid (Unilever, The Netherlands). The tested cellulase preparationswere ALKO4125, ALKO4179, ALKO4237 and ALKO4265 (Example 1) concentratedculture filtrates and purified 20K- and 50K-cellulases from the ALKO4237strain (Example 9).

Cellulase preparations were incubated at 40° C. in 0.25% detergentsolutions. The activity against hydroxyethylcellulose (ECU l ml,Example 1) was measured (pH 7, 50° C.) from samples taken after 5-30minutes incubation.

The tested preparations were as follows:

Culture filtrates:

ALKO4125: 780 ECU/ml (pH 7, 50° C.)

ALKO4179: 830 ECU/ml

ALKO4265: 760 ECU/ml

ALKO4237: 650 ECU/ml

Purified proteins:

20K-cellulase: 9423 ECU/ml

50K-cellulase: 10100 ECU/ml

The results are shown in Tables XI-XIII.

ALKO4179, ALKO4265 and ALKO4237 cellulase preparations and 20K- and50K-cellulases stay almost 100% stable for 30 minutes at 40° C. in allthree tested detergents. ALKO4125 stays stable for 30 minutes at 40° C.in Colour Detergent Liquid and in OMO® Neste.

TABLE XI Stability of different cellulases in 0.25% Colour DetergentLiquid (pH 7.5-7.9) enzyme dosage % of activity left preparation % (ml)pH* 0′ 5′ 10′ 20′ 30′ Culture filtrates: ALKO4125 6 7.3 100 97 98 98 99ALKO4179 6 7.1 100 99 100  100  10 ALKO4265 6 7.2 100 100  100  100 100  ALKO4237 6 7.1 100 100  82 95 100  Purified proteins from ALKO4237:20K-cellulase 1 7.8 100 98 99 97 100  50K-cellulase 1 7.6 100 100  100 100  100  *pH of the 0.25% detergent + enzyme solution after 30′incubation

TABLE XII Stability of different cellulases in 0.25% OMO ® Total (orOMO ® Neste pH 8.5) enzyme dosage % of activity left preparation % (ml)pH* 0′ 5′ 10′ 20′ 30′ Culture filtrates: ALKO4125 6 7.8 100 98 96 86 87ALKO4179 6 7.3 100 98 96 96 99 ALKO4265 6 7.1 100 100  100  100  100 ALKO4265 4 7.8 100 99 97 100  100  ALKO4237 4 7.8 100 100  100  99 100 ALKO4237 2 7.3 100 99 97 99 99 Purified proteins from ALKO4237:20K-cellulase 1 8.2 100 100  99 93 100  50K-cellulase 1 7.8 100 95 92 9594 *pH of the 0.25% detergent + enzyme solution after 30′ incubation

TABLE XIII Stability of different cellulases in 0.25% OMO ® Color (pH9.6-10) enzyme dosage % of activity left preparation % (ml) pH* 0′ 5′10′ 20′ 30′ Culture filtrates: ALKO4125 6 9.6 100 (15) (15) (13) (14)ALKO4179 6 8.3 100 97 100  97 99 ALKO4265 6 9.1 100 100  100  100  100 ALKO4265 4 8.5 100 93 95 99 98 ALKO4237 4 8.5 100 98 96 96 99 ALKO4237 29.1 100 93 95 99 98 Purified proteins from ALKO4237: 20K-cellulase 1 9.8100 99 100  100  100  50K-cellulase 1 8.9 100 100  100  100  100  *pH ofthe 0.25% detergent + enzyme solution after 30′ incubation

Example 14

Function of Neutral Cellulases in Detergents in HEC Substrate

The function of different neutral cellulases in detergents wasdetermined by using hydroxyethylcellulose (HEC) as a substrate. Thetested cellulase preparations were ALKO4265 and ALKO4237 concentratedculture filtrates and purified 20K- and 50K-cellulases from ALKO4237strain. HEC substrates were prepared by dissolving 1% HEC into 0.25%detergent solutions. By using these substrates the activity against HEC(ECU/ml) was measured at 40° C. from each cellulase preparations asdescribed in Example 1. Detergents and cellulase preparations used inthese experiments are described in Example 13.

pH of the substrates:

HEC/buffer pH 7 HEC/Colour Detergent Liquid pH 7.5 HEC/OMO ® Total pH7.8 HEC/OMO ® Color pH 9.7

TABLE XIV ECU of the cellulase preparations in different detergents(compared as % from the ECU activity measured in pH 7 buffer) Activity %ECU/ ECU/ ECU/ ECU/ col.det. OMO ® OMO ® preparation buffer liquid TotalColor culture filtrates: ALKO4265 100 89 96 59 ALKO4237 100 97 95 40purified proteins: 20K-cellulase 100 100 93 81 50K-cellulase 100 92 7946

ALKO4237 and ALKO4265 cellulase preparations and 20K- and 50K-cellulasesfunction in all three tested detergents when using HEC as a substrate.

Example 15

Use of Neutral Cellulases in Detergents on Cotton Woven Fabrics

In this experiment is described the ability of the neutral cellulases tofunction as fabric-softening agent and to prevent fizzing and thus toreduce pilling tendency from cotton fabric after repeated launderings indetergents. The tested cellulase preparations were ALKO4237 concentratedculture filtrate and the purified 20K- and 50K-cellulases from ALKO4237strain (Examples 1 and 9).

The washing experiment was carried out with a Launder-Ometer LP-2(Atlas, Ill., USA). About 10 g of prewashed (Example 3) unbleachedcotton woven fabric swatch was loaded into 1.2 liter container thatcontained 150 ml of 0.25% detergent solution with or without cellulase.Cellulase dosages were based on protein amounts. Detergent solutionswere OMO® Total (Lever, UK) and Colour Detergent Liquid (Unilever, TheNetherlands). A quantity of steel balls were added into each containerto increase the mechanical action. The Launder-Ometer was run at 42 rpmfor 0.5 or 1 hour at 40° C. The material was washed 4 times withintermediate rinsing and drying.

Weight loss (see Example 6) was used to describe the amount of fizzremoved from the fabrics surface.

TABLE XV Weight loss of the fabrics after the first washing time withneutral cellulases in detergents. enzyme dosage sample as protein/ timeweight loss no preparation g fabric h % In Colour Detergent Liquid: 1 —— 1 0.05 2 ALKO4237 11  1 0.3 3 ALKO4237 22  1 0.7 4 20K-cellulase 2 10.1 5 20K-cellulase 5 1 0.5 6 20K-cellulase 8 1 1.0 7 50K-cellulase 2 10.1 8 50K-cellulase 5 1 0.2 9 — — 0.5 0.2 10 20K-cellulase 8 0.5 0.5 InOMO ® Total: 11 — — 1 0.03 12 20K-cellulase 8 1 1.1 13 — — 0.5 0.1 1420K-cellulase 8 0.5 0.7

In the Table XV it is shown that after the first washing inLaunder-Ometer weight loss of the fabrics were increased clearly morewith cellulase treated fabrics than with the fabrics treated with thesole detergent Also weight loss was increased as a function of cellulasedosage and further with 20K-cellulase weight loss was increased whenwashing time was raised from 0.5 hour to 1 hour. 20K-cellulase workedequally well in Colour Detergent Liquid and in OMO® Total. These resultsindicate that particularly the 20K-cellulase and ALKO4237 cellulasepreparation function in detergents as fuzz removing agents after alreadyone wash time.

After three further washing times with samples 1, 2, 4 and 7 (Table XV)the evaluation of the fabrics was performed by a panel consisting ofthree persons. Panelists were asked to evaluate the softness and visualappearance of the treated fabrics as follows.

The softness of the fabrics:

A. the fabric treated with cellulase is softer than the fabric treatedwithout cellulase

B. the fabric treated with cellulase is as soft as the fabric treatedwithout cellulase

C. The fabric treated with cellulase is harder than the fabric treatedwithout cellulase

The results are shown in Table XVI.

Visual appearance of the fabrics was evaluated by ranking the fabrics ona score from 1 to 5. Score of 5 gave no fuzz or pills and the fabrictexture became more apparent. Score of 1 gave many pills and fuzz. Totalscore for each fabric was calculated and divided by the number of thepanelists. The average score of the visual appearance of each fabric isshown in Table XVI.

TABLE XVI Softness and visual appearance of the fabrics after 4 repeatedwashing times with neutral cellulases in detergents. enzyme dosage asprotein/ time visual preparation g fabric h softness appearance InColour Detergent Liquid: — — 1 1 ALKO4237 11  1 100%: softer 3.2 withcellulase 20K-cellulase 2 1 100%: softer 3.7 with cellulase50K-cellulase 2 1 100%: no difference 1.7

After the 4 treatments the cellulase treated fabrics had clearly bettervisual appearance than the fabrics that were treated with soledetergent. Thus fabrics treated with these cellulases maintained goodappearance and the fuzziness was prevented after repeated washingscompared to the fabric treated without cellulases. Also after 4 washtimes the ALKO4237 and 20K-cellulase treated fabrics were softer thanthe fabric treated with sole detergent.

Example 16

Use of Neutral Cellulases in Detergents on Cotton Fleecy Knit

In this experiment is described the ability of the neutral cellulases tofunction as fabric-softening agent and to prevent fuzzing and thus toreduce pilling tendency from coloured cotton fleecy knit after repeatedlaunderings in detergents. The tested cellulase preparations wereALKO4237 concentrated culture filtrate and the purified 20K-cellulasefrom ALKO4237 strain (Examples 1 and 9).

Green cotton fleecy knit swatches were washed at Launder-Ometer inColour Liquid Detergent or in OMO® Total for 1 h 3 or 10 times with orwithout cellulases as described in Example 15.

The evaluation of the knits was performed by a panel consisting of threepersons. Panelists were asked to evaluate the softness and visualappearance (both right and reverse sides) of the treated knits asdescribed in Example 15. Weight loss of the knits was determined asdescribed in Example 15. The results are shown in Table XVII.

After the 3 washing times the 20K-cellulase treated knits had bettervisual appearance both on the right and reverse side than the knitstreated with sole detergent. Knits treated 10 times with ALKO4237cellulase preparation had clearly better visual appearance and brightergreen colour than the knits treated only with detergent The bettervisual appearance of the cellulase treated knits was detected alreadyafter 1 wash time (especially on the reverse side) and it was furtherdeveloped during the additional washings. The cellulase treated knitswere also softer than the knits treated with sole detergent

TABLE XVII Softness, weight loss and visual appearance of the fleecyknits after 3 or 10 repeated washing times with or without cellulases indetergents. Before washings pH of the 0.25% Colour Detergent Solutionwas 7.9 and 8.4 of the 0.25% OMO ® Total solution. enzyme pH weightvisual appearance dosage as washing after loss right reverse preparationprotein/g fabric times washings % softness side Colour Detergent Liquid— —  3 7.9 0.46 1 1 20K*  5  3 7.4 0.88 33%: softer with cellulase 1.52.7 — — 10 8.0 1.46 1 1 A4237 20 10 7.9 2.80 100%: softer with cellulase2.5 2.8 OMO ® Total — — 10 8.3 0.57 1 1 A4237 20 10 8.2 1.57 100%:softer with cellulase 2.3 2.8 * = 20K-cellulase

Example 17

Use of neutral cellulases in detergents on aged cotton fleecy knit

In this experiment is described the ability of the neutral cellulases tofunction as fabric-renewal and -softening agent.

Green cotton fleecy knit was washed 10 times, with intermediate drying,in Cylinda washing machine with progranme 3 at 60° C., 10 ml of OMO®Color (Lever, UK). This was to simulate the washings of the knit inpractice.

After 10 treatments this aged knit had unattractive and faded appearancewith a lot of fuzz at the surface.

After these 10 repeated washes the fleecy knit was used for the washingexperiments with or without cellulase. Knit swatches were washed atLaunder-Ometer in Colour Liquid Detergent for 1 h 1 to 3 times asdescribed in example 15 with intermediate rinsing and drying. Thecellulase preparations used were ALKO4237 concentrated culture filtrateand purified 20K- and 50K-cellulases from ALKO4237 (Example 9).

The evaluation of the knits was performed by a panel consisting of threepersons. Panelists were asked to evaluate the softness and visualappearance (both right and reverse sides) of the treated knits asdescribed in Example 15. Weight loss of the knits was determined asdescribed in Example 15. The results are shown in Table XVIII.

After one wash time ALKO4237 and 20K-cellulase treated knits hadslightly beter visual appearance than the knit treated with soledetergent. The good visual appearance and more attractive look wasfurther developed to the 20K-cellulase treated knits after 2 and 3 washtimes. Visual appearance was also improved after two wash times on theknits treated with 50K-cellulase compared to the knit treated with soledetergent. As general, the knits treated with cellulases had clearlyimproved and attractive look while the knits treated without cellulasehad still unattractive and faded appearance.

TABLE XVIII Softness, weight loss and visual appearance of the agedfleecy knits after 1 to 3 repeated washing times with or withoutcellulases in detergents. Before washings pH of the 0.25% ColourDetergent Solution was 7.9. enzyme pH weight visual appearance dosage asmg washing after loss right reverse preparation protein/g fabric timeswashings % softness side — — 1 ND 0 1 1 ALKO4237 20 1 ND 0.61 100%: nodifference 1 1.5 20K*  5 1 ND 0 100%: no difference 1.5 1.5 — — 2 7.90.10 1 1 20K*  5 2 7.7 0.46 100%: softer with cellulase 2.5 2.2 50K*  52 7.7 0.26 100%: no difference 1 1.2 50K* 15 2 7.3 0.49 100%: nodifference 1 1.3 — — 3 ND 0.31 1 1 20K*  5 3 ND 0.88 100%: softer withcellulase 3.0 2.2 ND = not determined * = 20K- or 50K-cellulase

Example 18

Isolation of the ALKO4237 chromosomal DNA and construction of thegenomic library

Melanocarpus albomyces ALKO4237 was grown in shake flask cultures inpotato dextrose (PD; Difco, USA)- medium at 42° C., 250 rpm for 3 days.The chromosomal DNA was isolated according to Raeder and Broda, Lett.Appl. Microbiol. 1:17-20 (1985). Briefly, the mycelium was washed with20 mM EDTA and lysed in extraction buffer (200 mM Tris-HCl (pH 8.5), 250mM NaCl, 25 mM EDTA, 0.5% SDS). The DNA was extracted with phenol and amixture of chloroform:isoamyl alcohol (24:1 v/v). RNA was digested withRNase.

The chromosomal DNA was partially digested with Sau3A (BoehringerMannhein, Germany) and treated with calf intestine alkaline phosphatase.DNA ranging from 5-15 kb was isolated from an agarose gel usingbeta-agarase (Boehringer Mannheim, Germany) and used to construct thegenomic ALKO4237 library.

The predigested Lambda DASH®II BamHI Vector Kit (Stratagene, USA) wasused to construct the library and the instructions of the manufacturerwere followed in all the subsequent steps. Briefly, about 200 ng of thesize-fractionated DNA was ligated into 1 μg of DASH®II prepared arms,and packaged using Gigapack II packaging extract (Stratagene, USA). Thetiter of the library was determined by infecting E. coli XL1-Blue MRA(P2)-cells with serial dilutions of the packaged phage and plating onNZY plates. The library was stored at 4° C. in SM-buffer, with 4% (v/v)chloroform. It was used for screening without amplification.

Example 19

Amplification, cloning and sequencing of the 20K-cellulase DNA withdegenerate primers

To amplify the 20K-cellulase gene by polymerase chain reaction (PCR), apair of degenerate primers based on the peptide sequences (FIG. 17) (SEQID NOS:1-12) was synthesized. Primer 1 (429-32) (SEQ ID NO:38) wasderived from the amino acids #8-14 of the N-terminal peptide #429 (FIG.17) (SEQ ID NO:1), and primer 2 (fr28-16) (SEQ ID NO:39) was designed asthe antisense strand for the amino acids #2-8 of the peptide fr28 (FIG.17) (SEQ ID NO:11) . Additional EcoR1 restriction sites were added atthe 5′-termini to facilitate the cloning of the amplified fragment.

Primer I (429-32)(SEQ ID NO:38)          EcoRI 5′ - ATA GAATTC TA(C/T)TGG GA(C/T) TG(C/T) TG(C/T) AA(A/G) CC                Y      W   D       C       C       K        P Primer2(fr28-16) (SEQ ID NO:39)          EcoRI 5′ - ATA GAATTC TT (A/G)TC(A/C/G/T)GC (A/G)TT (C/T)TG (A/G)AA                N       D           A       N       Q      F CCA  W

In the PCR reaction, 1 μg of the purified ALKO4237 genomic DNA (Example18) was used as the template. Dynazyme DNA polymerase (Finnzymes Ltd,Finland) was used according to the supplier's instructions.

Template DNA (0.7 μg/μl) 1.4 μl Primer 1 (0.5 μg/μl) 1 μl Primer 2 (0.5μg/μl) 1 μl dNTPs (2 mM) 5 μl 10 × PCR buffer 10 μl dH2O 82 μl Dynazyme(2 U/μl) 1 μl Total 101.4 μl

The PCR reaction was performed under the following conditions:

Step 1 95° C. 5 min Step 2 95° C. 1 min Step 3 56° C. 1 min Step 4 72°C. 1 min Step 5 go to “step 2” 29 more times Step 6 72° C. 8 min Step 7 4° C. hold

Ten μl of reaction mixture was analyzed by agarose gel electrophoresis,and a single band corresponding to about 600 bp in length was detected.The remaining of the PCR product was digested with EcoR1 restrictionendoglucanase, and run by agarose electrophoresis. The agarose sectioncontaining the DNA fragment was excised, and purified by the Magic PCRPreps (Promega, USA) method according to supplier's instructions. Theisolated fragment was ligated with pBluescript II SK+ (Stratagene, USA)plasmid which was cut similarly with EcoR1. Competent Escherichia coliXL-Blue cells (Stratagene, USA) were transformed with the ligationmixture. Plasmid DNA from a few of the resulting colonies was isolatedby the Magic Minipreps (Promega, USA) method according to supplier'sinstructions. The plasmid DNA was analyzed by agarose electrophoresis,and one clone with expected characteristics was designated pALK549.

The Melanocarpus DNA from pALK549 was sequenced by using ABI (AppliedBiosystems, USA) kits based on fluorescent-labeled T3 and T7 primers, orsequence-specific primers with fluorescent-labeled dideoxynucleotides bythe Taq dye primer cycle sequencing protocol in accordance with thesupplier's instructions. Because of high GC content of the MelanocarpusDNA, the sequencing reactions were performed at annealing temperature of58° C., with 5% (v/v) DMSO. Sequencing reactions were analyzed on ABI373A sequencer (Applied Biosystems, USA), and the sequences obtainedwere characterized by using the Genetics Computer Group SequenceAnalysis Software Package, version 7.2.

The insert (594 bp) in pALK549 was found to encode the majority of the20K-cellulase derived peptides (FIG. 17) (SEQ ID NOS:1-12). The PCRamplified DNA (in addition to the primers) corresponds to thenucleotides 175-716 in FIG. 19(A and B) (SEQ ID NO:30).

Chromosomal DNA from Myriococcum sp. ALKO4124 was isolated as describedin Example 18. A PCR reaction with the primers 429-32 and fr28-16 andALKO4124 chromosomal DNA as the template produced a fragment of samesize as from ALKO4237 DNA. This fragment was partly sequenced, and wasalmost identical to the ALKO4237 sequence. It is concluded thatMyriococcum sp. ALKO4124 has a protein, which is almost identical to the20K-cellulase of Melanocarpus albomyces ALK04237. This result is also inagreement with the observation that the ALK04237 20K-cellulase specificantibodies also recognize a 20K protein band from ALKO4124 growth mediumin Western analysis (FIG. 14). Enzymes from both stnnns gave similargood results in biostoning experiments (Examples 3 and 4).

Example 20

Cloning and sequencing the Melanocarpus albomyces ALK04237 20K-cellulasegene

E. coli XL1-Blue MRA (P2) -cells (Stratagene, USA) were grown in LB+0.2%maltose+10 mM MgSO₄, and diluted to OD₆₀₀=0.5. The cells were infectedwith the Melanocarpus albomyces ALKO4237 genomic library (Example 18)for 15 min at 37° C., and plated with NZY top agar on the NZY plates.Plates were incubated at 37° C. overnight. The plaques were transferredonto a nylon filter (Hybond, Amersham, UK) according to Stratagene'sinstructions.

The purified PCR fragment (Example 19) was labeled with digoxigeninaccording to Boehringer, DIG DNA Labeling and Detection Nonradioactive,Application Manual. Hybridization was performed at 68° C. The positiveclones were picked in SM buffer/chloroform, and purified with a secondround of screening.

Under these conditions 4 positive clones were found. The large scalebacteriophage lambda DNA isolation from the clones was done according toSambrook et al., in Molecular Cloning: A Laboratory Manual, 2nd edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Thephage DNAs were analyzed by digestion of the DNA with severalrestriction enzymes, and the digested DNA was hybridized with thePCR-probe. Three hybridizing frgments were isolated: about 2.6 kbEcoR1-XhoI fragment, about 4.9 kb XhoI fragment and about 3 kb SacIfragment. These were inserted into similarly cut pBluescript II SK+vector (Stratagene, USA), creating plasmids pALK1221, pALK1222 andpALK1223, respectively (FIG. 18).

The Melanocarpus albomyces DNA in pALK1221 was sequenced as described inExample 19. The DNA sequence encoding the Melanocarpus albomyces20K-cellulase is shown in FIG. 19(A and B) (SEQ ID NO:30). The sequenceis 936 bp in length, and has an open reading frame (ORF) coding for 235amino acids; the gene has two introns. The putative signal peptideprocessing site is after alanine-21, and the N-terminus of the matureprotein begins at alanine-22, as suggested by the peptide sequencingresults (FIG. 17, peptide #429(SEQ ID NO:1)). The ORF predicts a proteinwith a molecular weight of 25.0 kDa for the full-length preprotein, and22.9 kDa for the mature protein. This is in good agreement with theresults obtained from the protein purification work (Example 10). Theseresults also verify that the about 35 kDa protein detected previouslywith the 20K-cellulase antiserum (Example 10) is a different geneproduct than the 20K-cellulase.

The 20K-cellulase of Melanocarpus albomyces appears to belong to familyK of cellulases and family 45 of glycosyl hydrolases (Henrissat &Bairoch, Biochem. J. 293:781-788 (1993)). The 20K-cellulase showshomology (about 76% identify in 235 amino acid overlap) towards theHumicola insolens endoglucanase V (embl:a23635), but the 20K-cellulasehas the surprising feature that it does not harbor the cellulose bindingdomain (CBD) and its linker, which are characteristic of the Humicolainsolens endoglucanase V and other related endoglucanases (Schülein etal., 1993, In: Suominen & Reinikainen (eds), Foundation for Biotechnicaland Industrial Fermentation Research, Helsinki, vol. 8, 109.; Saloheimoel al., 1994, Mol. Microbiol 13, 219). This feature of the 20K-cellulasemay account for the excellent performance of the enzyme in biostoningexperiments (Example 10).

Example 21

Amplification, cloning and sequencing of 50K-cellulase DNA withdegenerate primers

The peptides derived from the 50K-cellulase (Table IX) shared somehomology towards Humicola grisea endoglucanase I (DDBJ:D63516). Toamplify the 50K-cellulase gene by polymerase chain reaction (PCR) a pairof degenerate primers based on the peptide sequences (Table IX) (SEQ IDNOS:13-24) was synthetized Primer 1 (507-128) (SEQ ID NO:40) was derivedfrom the amino acids #5-10 of the peptide #507 (Table IX) (SEQ IDNO:13), and primer 2 (509-rev) (SEQ ID NO:41) was designed as theantisense strand for the amino acids #4-9 of the peptide 509 (Table IX)(SEQ ID NO:14). The order of the two peptides in the protein—and thecorresponding sense-antisence nature of the primers—was deduced fromcomparison with the Humicola grisea endoglucanase I.

Primer 1(507-128)(SEQ ID NO:40) 5′ - GA(C/T) GA(A/G) AC(A/C/G/T) GA(A/G)CA(C/T) (A/C)G      D        E       T           E       H       RPrimer 2(509-rev) (SEQ ID NO:41) 5′ -TA (A/C/G/T)GC (A/C/G/T)CC(A/C/G/T)CC (A/C/G/T)GG (A/G)TT      Y          A            G           G           P       N

In the PCR reaction, 1.5 μg of the purified ALKO4237 genomic DNA(Example 18) was used as the templete. Dynazyme DNA polymerase(Finnzymes Ltd, Finland) was used according to the supplier'sinstructions.

Template DNA (0.3 μg/μl) 5 μl Primer 1 (0.5 μg/μl) 1 μl Primer 2 (0.5μg/μl) 1 μl dNTPs (2 mM) 5 μl 10 × PCR buffer 10 μl dH2O 79 μl Dynazyme(2 U/μl) 1 μl Total 102 μl

The PCR reaction was performed under the following conditions:

Step 1 95° C. 5 min Step 2 95° C. 1 min Step 3 56° C. 1 min Step 4 72°C. 1 min Step 5 go to “step 2” 29 more times Step 6 72° C. 8 min Step 7 4° C. hold

Ten μl of reaction mixture was analyzed by agarose gel electrophoresis,and a single band corresponding to about 160 bp in length was detected.The remaining of the PCR product was loaded on a agarose gelelectrophoresed, and the agarose section containing the DNA fragment wasexcised, and purified by the Magic PCR Preps (Promega, USA) methodaccording to the supplier's instructions.

The isolated fragment was ligated with pBluescript II SK+ (Stratagene,USA) plasmid which had been digested with EcoRV endonuclease, andddT-tailed as described in Holton and Graham (1990) Nucl. Acids Res. 19,1156. Competent Escherichia coli XL-Blue cells (Statagene, USA) weretransformed with the ligation mixture. Plasmid DNA from a few of theresulting colonies was isolated by the Magic Minipreps (Promega, USA)method according to the supplier's instructions. The plasmid DNA wasanalyzed by agarose electrophoresis, and one clone with expectedcharacteristics was designated pALK1064.

The insert (161 bp) in pALK1064 was sequenced as described in Example19, and was found to contain an ORF, which predicted a peptidehomologous to Humicola grisea endoglucanase I (DDBJ:D63516). The ORFalso encoded the peptide #612 (Table IX) (SEQ ID NO:21) from thepurified 50K-cellulase. The PCR amplified DNA (in addition to theprimers) corresponds to the nucleotides 404-530 in FIG. 21 (SEQ IDNO:32).

PCR with the primers 507 and 590-rev with ALKO4124 chromosomal DNA astemplate (Example 19) produced a fragment of same size as from ALKO4237DNA. This suggests that Myriococcum sp. ALKO4124 has a protein verysimilar to the 50K-cellaulase of Melanocarpus albomyces ALKO4237. Thisis also supported by the fact that enzymes from both strains gavesimilar good results in biostoning experiments.

Example 22

Cloning and sequencing the Melanocarpus albomyces ALKO4237 50K-cellulasegene

The genomic bank of Melanocarpus albomyces ALKO4237 was prepared forhybridization as described in Example 20. The purified PCR fragmentcarrying part of the 50K-cellulase gene (Example 21) was labeled withdigoxigenin according to Boehringer, DIG DNA Labeling and DetectionNonradioactive, Application Manual. Hybridization was performed at 68°C. The positive clones were picked in SM buffer/chloroform, and purifiedwith a second round of screening.

Under these conditions 10 positive clones were found. The large scalebacteriophage lambda DNA isolation from the clones was done according toSambrook el al., 1989. The phage DNAs were analyzed by digestion of theDNA with several restriction enzymes, and the digested DNA washybridized with the 50K-cellulase-specific PCR-probe. Four hybridizingfragments were isolated: about 2.8 kb SacI-Xhol fragment, about 5 kbSacI fragment, about 3.2 kb Xhol fragment, and about 2 kb EcoR1fragment. These were inserted into similarly cut pBluescript II SK+vector (Stratagene, USA), creating plasmids pALK1234, pALK1233, pALK1226and pALK1227, respectively (FIG. 20).

The Melanocarpus albomyces ALKO4237 DNA was sequenced from the50K-cellulase specific plasmids mentioned above. The sequencing protocolhas been described in Example 19.

The DNA encoding the Melanocarpus albomyces 50K-cellulase is shown inFIG. 21 (A, B and C) (SEQ ID NO:32). The sequence reveals an ORF ofabout 1363 bp in length, interrupted by one intron. The ORF codes for428 amino acids. The predicted protein has a molecular weight of 46.8kDa and after signal peptide cleavage of 44.8 kDa. All the peptides inTable IX (SEQ ID NOS:13-24) are found in the predicted protein sequence(FIG. 2) (FIG. 4) (SEQ ID NO:33), although some amino acids identifiedwith uncertainty during the peptide sequencing proved to be incorrect.The protein shows homology to Humicola grisea endoglucanase I(DDBJ:D63516).

Example 23

Amplifcation, cloning and sequencing of 50K-cellulase B DNA withdegenerate primers

The peptides derived from the 50K-cellulase B (Table X) (SEQ IDNOS:25-29) shared some homology towards Humicola griseacellobiohydrolase I (DDBJ:D63515). To amplify the 50K-cellulase B geneby polymerase chain reaction (PCR) a pair of degenerate primers based onthe peptide sequences (Table X) (SEQ ID NOS:25-29) was synthesized.Primer 1 (636) was derived from the amino acids #1-5 of the peptide #636SEQ ID NO:42 (Table X) (SEQ ID NO:24) (the first amino acid was guessedto be lysine, because this peptide was isolated after digestion with aprotease cleaving after lysines), and primer 2 (534-rev) (SEQ ID NO:43)was designed as the antisense strand for the amino acids #3-8 of thepeptide #534 (Table X) (SEQ ID NO:25). The order of the two peptides inthe protein—and the corresponding sense-antisense nature of theprimers—was deduced from comparison with the Humicola griseacellobiohydrolase I.

Primer 1(636) (SEQ ID NO:42) 5′ - AA(A/G) CA(C/T) GA(A/G) TA(C/T)GG(A/C/G/T) AC       K      H        E       Y       G           TPrimer 2 (534-rev) (SEQ ID NO:43) 5′- CC (A/G)TA (A/G)AA (A/G)TC(A/C/G/T) GG (A/G)TT      G       Y       F       D            P       N

In the PCR reaction, 1.5 μg of the purified ALKO4237 genomic DNA(Example 18) was used as the template. Dynazyme DNA polymerase(Finnzymes Ltd, Finland) was used according to the supplier'sinstructions.

Template DNA (0.3 μg/μl) 5 μl Primer 1 (0.3 μg/μl) 1.7 μl Primer 2 (0.3μg/μl) 1.7 μl dNTPs (2 mM) 5 μl 10 × PCR buffer 10 μl dH2O 80 μlDynazyme (2 U/μl) 1 μl Total 104.4 μl

The PCR reaction was performed under the following conditions:

Step 1 95° C. 5 min Step 2 95° C. 1 min Step 3 48° C. 1 min Step 4 72°C. 2 min Step 5 go to “step 2” 34 more times Step 6 72° C. 8 min Step 7 4° C. hold

Twenty μl of reaction mixture was analyzed by agarose gelelectrophoresis, and a few bands were detected. One of the bands had anapparent size of 700 bp, which size was in agreement with size one wouldexpect, when comparing with Humicola grisea cellobiohydrolase gene,particularly, if the fragment contained one or more introns. The PCRproducts were purified by the Magic PCR Preps (Promega, USA) methodaccording to the supplier's instructions.

The isolated fragments was ligated with pBluescript II SK+ (Stratagene,USA) plasmid which had been digested with EcoRV endonuclease, andddT-tailed as described in Holton and Graham, Nucl. Acids Res. 19:1156(1990). Competent Escherichia coli XL-Blue cells (Stratagene, USA) weretransformed with the ligation mixture. Plasmid DNA from a few of theresulting colonies was isolated by the Magic Minipreps (Promega, USA)method according to the supplier's instructions. The plasmid DNA wasanalyzed by agarose electrophoresis, and one clone with about 700 bpinsert was designated pALK1224.

The insert in pALK 1224 was sequenced as described in Example 19, andwas found to contain an ORF encoding the whole peptide #636 (SEQ IDNO:29) from the 50K-cellulase B (Table X). The ORF predicted a peptidehomologous to Humicola grisea cellobiohydrolase I (DDBJ:D63515). The PCRamplified DNA (in addition to the primers) corresponds to thenucleotides 371-1023 in FIG. 23(A, B and C) (SEQ ID NO:34).

Example 24

Cloning and sequencing the Melanocarpus albomyces ALKO4237 50K-cellulaseB gene

The genomic bank of Melanocarpus albomyces ALKO4237 was prepared forhybridization as described in Example 20. The insert in pALK1224 wasremoved by digesting the plasmid with restriction endoglucanases EcoRIand HindIII. The digested plasmid DNA was run by agaroseelectrophoresis. The agarose section containing the about 700 bp DNAfragment was excised, and purified by the Magic PCR Preps (Promega, USA)method according to the supplier's instructions.

The purified PCR fragment from pALK1224 carrying part of the50K-cellulase B gene (Example 23) was labeled with digoxigenin accordingto Boehringer, DIG DNA Labeling and Detection Nonradioactive,Application Manual. Hybridization was performed at 68° C. The positiveclones were picked in SM buffer/chloroform, and purified with a secondround of screening.

Under these conditions 3 positive clones were found. The large scalebacteriophage lambda DNA isolation from the clones was done according toSambrook et al., in Molecular Cloning A Laboraiory Manual, 2nd edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Thephage DNAs were analyzed by digestion of the DNA with severalrestriction enzymes, and the digested DNA was hybridized with the50K-cellulase B specific PCR probe. A hybridizing 3.5 kb NotI fragmentwas isolated, and inserted into similarly cut pBluescript II SK+ vector(Stratagene, USA), creating plasmid pALK1229 (FIG. 22).

The extreme 5′-end of the gene was found by hybridizing the phage DNAswith 0.2 kb NotI-PstI-fragment from pALK1229. A hybridizing 2.4 kbPstI-fragment was isolated and inserted into similarly cut pBluescriptII SK+ vector (Stratagene,USA), creating plasmid pALK1236 (FIG. 22).

Part of the inserts in pALK1229 and pALK1236 were sequenced as describedin Example 19. The DNA encoding the Melanocarpus albomyces 50K-cellulaseB is shown in FIG. 23 (A, B and C) (SEQ ID NO:34). The sequence revealsan ORF of 1734 bp in length interrupted by five introns. The ORF codesfor 452 amino acids. The predicted protein has a molecular weight of49.9 kDa and after signal peptide cleavage of 47.6 kDa. All the peptidesin Table X (SEQ ID NOS:25-29) are found in the predicted proteinsequence (FIGS. 23 A, B and C) (SEQ ID NO:35), although some amino acidsidentified with uncertainty during the peptide sequencing proved to beincorrect. The predicted protein shows homology to Humicola griseacellobiohydrolase I (DDBJ:D63515) and other cellobiohydrolases. However,50K-cellulase B has the surprising feature that it does not harbor thecellulose binding domain (CBD) and its linker, which is characteristicto Humicola grisea cellobiohydrolase I and many othercellobiohydrolases.

Example 25

Screening the Melanocarpus albomyces ALKO4237 genomic library withTrichoderma reesei celulases genes

The genomic bank of Melanocarpus albomycesALKO4237 was prepared forhybridization as described in Example 20.

A DNA fragment carrying Trichoderma reesei cbh1 specific DNA wasisolated by cutting plasmid pTTc01 (FIG. 24) with restrictionendonuclease HincII, and isolating the about 1.6 kb fgent from agarosegel after electrophoresis. A DNA fragment carrying Trichoderma reeseiegl2 specific DNA was isolated by cutting plasmid pMS2 (FIG. 25) withrestriction endonucleases BamHI and EcoRI, and isolating the about 1.5kb fragment from agarose gel after electrophoresis. The cloning of thecbh1 gene is described in Teeri et al., Bio/Technology 1:696-699 (1983)and the DNA sequence is described in Shoemaker et al., Bio/Technology 1:691-696 (1983). The egl2 (originally called “egl3”) gene is described inSaloheimo et al., Gene 63:11-21 (1988).

The fragments were labeled with digoxigenin according to Boehringer, DIGDNA Labeling and Detection Nonradioactive, Application Manual.Hybridization was performed at 68° C. with the cbh1 probe and at 60 ° C.with the egl2 probe. The positive clones were picked in SMbuffer/chloroform, and purified with a second round of screening.

Under these conditions 13 cbh1 positive and 6 egl2 positive clones werefound. One clone hybridized to both probes. The lambda DNA was isolatedfrom the clones as described above. The phage DNAs were analyzed bydigestion of the DNA with several restriction enzymes, and the digestedDNA was hybridized with the cbh1 and egl2 probes. The clones were alsohybridized with the 20K-cellulase-specific PCR fragment (Example 19).One clone (lambda-16) was clearly positive, and two other clones(lambda-8/1 and lambda-5/2) were weakly positive; all these clones wereoriginally picked with the cbh1 probe.

An about 4 kb EcoRI fragment from lambda-16, which hybridized to boththe Trichoderma reesei cbh1 probe and to the 20K-cellulase specific PCRfragment, was isolated from agarose gel after electrophoresis, andinserted into similarly cut pBluescript II SK+. The resulting plasmidwas named pALK1230 (FIG. 26).

Part of the insert in pALK1230 was sequenced as described in Example 19.The DNA appears not to encode the 20K-cellulase, but codes for a proteinhomologous to several cellulases, particularly at the cellulose bindingdomain (CBD) area. Thus the gene product very likely has high affmitytowards cellulosic material, and therefor this gene product wasdesignated as protein-with-CBD. The sequence is shown in FIG. 27 (SEQ IDNO:36).

PCR reactions with the primers 636 (SEQ ID NO:42) and 534-rev (SEQ IDNO:43) (Example 23) were performed with the DNA from the 19 lambdaclones as templates. One lambda clone, lambda-3, gave a band about 700bp in size, similar to that in Example 23 when ALKO4237 chromosomal DNAwas used as a template. This clone had originally been picked by theTrichoderma cbh1 probe. The lambda DNA was digested with severalrestriction endonucleases, and hybridized with the 50K-cellulase Bspecific probe. The clone showed similar restriction enzyme pattern asthe 3 clones in Example 24. It is concluded that lambda-3 also carriesthe 50K-cellulase B gene.

Example 26

Fusion proteins

A recombinant vector encoding the 20K-cellulase, 50K-cellulase or the50K-cellulase B is prepared by fusing the cellulase encoding sequencewith the sequence of Trichoderma reesei cellulase or hemicellulase or atleast one functional domain of said cellulase or hemicellulase, asdescribed in U.S. Pat. No. 5,298,405, WO 93/24621 and in Genbanksubmission L25310, incorporated herein by reference. Especially, theenzyme is selected from the group consisting of CBHI, CBHII, EGI, EGII,XYLI, XYLII and MANI, or a domain thereof, such as the secretion signalor the core sequence.

Fusion proteins can be constructed that contain an N-terminal mannanaseor cellobiohydrolase or endoglucanase core domain or the core and thehinge domains from the same, fused to one of the Melanocarpus cellulasesequences. The result is a protein that contains an N-terminal mannanaseor cellobiohydrolase or endoglucanase core or core and hinge regions,and a C-terminal Melanocarpus cellulase. The fusion protein containsboth the Trichoderma mannanase or cellobiohydrolase or endoglucanase andthe Melanocarpus cellulase activities of the various domains as providedin the fusion construct. Alternatively, mutations that modify theactivities of the Trichoderma mannanase or cellobiohydrolase orendoglucanase, or the Melanocarpus cellulase activities, may be includedin the constructions. In this case, the fusion proteins contain both themodified Trichoderma enzyme activity and the Melanocarpus cellulaseactivity of the various domains as provided in the fusion construct.

Fusion proteins can also be constructed such that the mannanase orcellobiohydrolase or endoglucanase tail or a desired fragment thereof,is placed before one of the Melanocarpus cellulase sequences, especiallyso as to allow use of a nonspecific protease site in the tail asprotease site for the recovery of the Melanocarpus cellulase part fromthe expressed fusion protein. Alternatively, fusion proteins can beconstructed that provide for a protease site in a synthetic linker thatis placed before one of the Melanocarpus cellulases, with or without thetail sequences.

Example 27

Hosts

The recombinant construct encoding the desired fusion proteins orMelanocarpus proteins are prepared as above, and transformed into afilamentous fungus such as Aspergillus spp., preferably Trichoderma spp.

Example 28

Trichoderma background for 20K-cellulase production

In this example is described stone-washing experiments to determine themost suitable background of Trichoderma cellulases for 20K-cellulaseproduction. The purpose of these experiments was to determine whichTrichoderma cellulases would cause backstaining in stone-washing atneutral conditions.

Trichoderma reesei strain ALKO3620 (endoglucanase 2 gene is deleted) waschosen as host for these experiments. In previous studies TrichodermaEGII (endoglucanase II) enzyme has been shown to cause detrimentaleffects to cotton fibre structures and thus to weaken the strengthproperties of cotton-containing fabrics (In: Miettinen-Oinonen et al.:Effects of cellulases on cotton fiber and fabrics. In: Proceedings ofthe TIWC96 Conference, 1996, Vol.1 (2), pp. 197.).

Stone-washing experiments were performed at pH 6.5 and 7 as described inExample 3 except that no Berol was used.

The tested Trichoderma cellulase preparations were:

ALKO3133 (egl2 and cbh2 deleted)

ALKO3269 (egl2 and egl1 deleted)

ALKO3268 (egl2 and cbh1 deleted)

The dosage of Trichoderma preparations was about 2.5 mg (=low dosage, L)or about 5 mg (=high dosage, H) of total protein per g of fabric. 0.4 mgof purified 20K-cellulase per g of fabric was used when needed.

Results of color measurements of treated denim fabrics are shown inTable XIX.

The stone-washing results show that ALKO3269 (egl2 and egl1 deleted)background causes less backstaining at neutral conditions than ALKO3268(egl2 and cbh1 deleted) or ALKO3133 (egl2 and cbh2 deleted) background.Thus the preferred host for 20K-cellulase production for biostoning isan ALKO3269-like strain. Although with higher 20K-cellulaseconcentrations the Trichoderma background has probably only very minorimportance. An ALKO3269-like background is probably as good for50K-cellulase and 50K-cellulase B production for biostoning as it is for20K-cellulase production.

TABLE XIX Color measurements of denim fabrics treated with differentTrichoderma cellulase preparations with (+) or without (−) 20K-cellulasepreparation/ 20K Right side Reverse side dosage +/− pH L b delta E L bdelta E — − 6.5 2.2 1.1 3.1 0.7 0.1 1.4 ALKO3620/L − 6.5 2.2 2.6 3.0−0.7 2.6 2.9 ALKO3620/L + 6.5 5.5 4.0 7.7 −1.3 5.0 5.5 ALKO3133/L − 6.51.9 2.2 3.7 0.2 1.6 2.3 ALKO3133/H − 6.5 4.2 1.9 4.5 −1.5 3.3 4.8ALKO3133/L + 6.5 5.7 4.3 7.8 0.3 4.5 5.0 ALKO3133/H + 6.5 8.5 4.0 9.4−1.4 5.9 7.8 ALKO3269/L − 6.5 2.9 1.9 4.4 0.8 0.8 1.6 ALKO3269/H − 6.54.3 1.5 4.5 0.6 1.3 2.6 ALKO3269/L + 6.5 6.6 4.2 8.7 1.1 4.0 4.3ALKO3269/H + 6.5 7.9 3.9 8.5 0.7 3.7 5.1 ALKO3268/L − 6.5 2.9 1.7 3.70.1 1.8 3.0 ALKO3268/H − 6.5 4.2 2.0 4.3 −0.7 3.4 5.0 ALKO3268/L + 6.55.9 3.2 7.7 −1.2 4.5 6.0 ALKO3268/H + 6.5 7.1 3.7 7.7 −2.0 5.8 7.3 — −7.0 2.9 0.8 2.6 0.7 0.5 1.5 ALKO3620/L − 7.0 3.3 1.2 1.9 1.7 0.3 1.1ALKO3620/L + 7.0 6.7 3.4 5.6 1.1 3.2 2.9 ALKO3133/L − 7.0 3.2 1.0 1.40.6 0.6 0.9 ALKO3133/L + 7.0 5.9 3.7 5.5 0.1 4.3 3.1 ALKO3269/L − 7.03.6 1.2 2.2 1.3 −0.3 1.3 ALKO3269/L + 7.0 6.4 3.4 5.9 1.2 3.2 2.8ALKO3268/L − 7.0 2.9 1.4 3.9 0.5 0.4 2.5 ALKO3268/L + 7.0 8.4 3.1 9.61.1 3.5 4.6

Example 29

Production of Melanocarpus albomyces ALKO4237 20K-cellulose in T. reesei

The Trichoderma reesei strains were constructed for Melanocarpusalbomyces ALKO4237 20K-cellulase production. Strains produceMelanocarpus 20K-cellulase and are unable to produce T. reesei'sendoglucanase II and cellobiohydrolase I or endoglucanase I. Suchpreparations deficient in Trichoderma cellulolytic activity, and themaking of same by recombinant DNA methods, are described in U.S. Pat.No. 5,298,405 or Suominen et al. (1993) High frequency one-step genereplacement in Trichoderma reesei. II. Effects of deletions ofindividual cellulase genes. Mol. Gen. Genet. 241: 523., incorporatedherein by reference.

In construction of the Melanocarpus albomyces 20K-cellulase producingstrains, the parental Trichoderma reesei strain ALKO3620 was transformedwith the expression cassettes from the plasmid pALK1231 or pALK1235(FIGS. 28 and 29). In the cassettes 20K-cellulase is expressed from thestrong cbh1 promoter. The integration of the expression cassettesresulted in the replacements of the parental cbh1 (pALK1231) or the egl1(pALK1235) genes.

In the host strain ALKO3620 the egl2 gene has been replaced by the 3.3kb XbaI-BglII fragment of the ble gene from Streptoalloteichushindustanus (Mattean et al. (1988) A vector of Aspergillustransformation conferring phleomycin resistance. Fungal Genet. Newslett.35: 25.; Drocourt et al. (1990) Cassettes of the Streptoalloteichushindustanus ble gene for transformation of lower and higher eukaryotesto phleomycin resistance. Nucl. Acids Res. 18:4009.) using therecombinant DNA methods described in U.S. Pat. No. 5,298,405,incorporated herein by reference.

The plasmids pALK1231 and pALK1235 that were used in the construction ofthe Melanocarpus cellulase producing strains are identical to each otherwith respect to cbhI promoter, 20K-cellulase gene and cbh1 terminatorwhich are described below:

T. reesei cbh1 (cellobiohydrolase 1) promoter: The promoter is fromTrichoderma reesei VTT-D-80133 (Teeri et al. (1983) The molecularcloning of the major cellulase gene from Trichoderma reesei.Bio/Technology 1: 696.). The 2.2 kb EcoRI - SacII fragment (Karhunen etal. (1993) High frequency one-step gene replacement in Trichodermareesei. I. Endoglucanase I overproduction. Mol. Gen. Genet. 241: 515.)was used in the construct. The sequence of the promoter area preceedingthe ATG was published by Shoemaker et al. (1983) Molecular cloning ofexo-cellobiohydrolase from Trichoderma reesei strain L27.Bio/Technology 1. 691.). The last 15 nucleotides of the T. reesei L27cbh1 promoter (the SacII site is underlined) are CCGCGGACTGGCATC (SEQ IDNO:44) (Shoemaker et al. 1983). The cbh1 promoter from the T. reeseistrain VTT-D-80133 has been sequenced at Alko Research Laboratories, andan one nucleotide difference in the DNA sequence has been noticed withinthe above mentioned region. In the T. reesei strain VTT-D-80133 thesequence preceeding the ATG is CCGCGGACTG/C/GCATC (SEQ ID NO:45) (theSacII site is underlined, the additional cytosine in the DNA sequence isbetween the slashes).

The nucleotides missing from the promoter (10 bps after the SacII to theATG) were added and the exact promoter fulsion to the first ATG of theMelanocarplus 20K-cellulase (see below) was done by using the PCR(polymerase chain reaction) method. The fusion and the PCR fragment weresequenced to ensure that no errors had occurred in the reaction. InpALK1231 the promoter area is also functioning as a homologous DNA(together with the cbh1 3′-fragment; see below) to target theintegration of the transforming DNA into the cbh1 locus.

Melanocarpus albomyces 20K-cellulase gene: The nucleotide sequence anddeduced amino acid sequence of the 20K-cellulase gene encoding an 20 kDacellulase is presented in Example 20 (FIG. 19) (SEQ ID NOS:30-31). A 0.9kb fragment beginning from ATG-codon was used in both plasmids.

Treesei cbh1 terminator: The 739 bp AvaII fragment (Karhunen et al.(1993) High frequency one-step gene replacement in Trichoderma reesei.I. Endoglucanase I overproduction. Mol. Gen. Genet. 241: 515.) starting113 bp before the STOP codon of the cbh1 gene was added after the20K-cellulase gene to ensure termination of transcription.

In addition the material described above the plasmid pALK1231 contains:

amdS gene: The gene has been isolated from Aspergillus nidulansVH1-TRSX6 and it is coding for acetamidase (Hynes et al. (1983)Isolation of genomic clones containing the amdS gene of Aspergillusnidulans and their use in the analysis of the structural and regulatorymutations. Mol. Cell. Biol. 3:1430.). Acetamidase enables the strain togrow by using acetamide as the only nitrogen source and thischaracteristics has been used for selecting the transformants. The 3.1kb fragment (SpeI - XbaI) from the plasmid p3SR2 (Kelly J. and Hynes M.(1985) Transformation of Aspergillus niger by the amdS gene ofAspergillus nidulans. EMBO J. 4: 475.) is used in the plasmids. Thefragment contains 1007 bps of the promoter area, 1897 bps of the codingregion (introns included) and the 183bps terminator area of the amdSgene.

cbh1 3′-fragment: The fragment was isolated from T. reesei ALKO2466 byusing plasmid rescue (1.7 kb, BamHI - EcoRI, starting 1.4 kb after thegene's STOP, Suominen et al. (1993) High frequency one-step genereplacement in Trichoderma reesei. II. Effects of deletions ofindividual cellulase genes. Mol. Gen. Genet. 241: 523.). Strain ALKO2466derives from the strain ALKO233 (Harkki et al. (1991) Geneticengineering of Trichoderma to produce strains with novel cellulaseprofiles. Enzyme Microb. Technol. 13: 227.). 3′-fragment is usedtogether with the promoter area to target the 20K-cellulase gene to thecbh1 locus by homologous recombination.

The plasmid pALK1235 contains:

hph gene: The gene encoding HmB phosphotransferase is originallyisolated from E. coli K-12 JM109 (Yanish-Perron et al. (1985) ImprovedM13 phage cloning vectors and host strains: nucleotide sequences of theM13mp18 and pUC19 vectors. Gene 33: 103.) and it confers resistance tohygromycin B (HmB). Resistance to hygromycin (inactivated byphosphorylation by HmB phosphotransferase) was used for selecting thetransformants. The hph gene together with the pki promoter and cbh2terminator (see below) is isolated from plasmid pRLM_(ex)30 (Mach el al.(1994) Transformation of Trichoderma reesei based on hygromycin Bresistance using homologous expression signals. Curr. Genet. 25: 567.)as a 2.2 kb NotI-PvuII fragment.

pki promoter: The about 0.75 kb pki (pyruvate kinase) promoter forexpressing hph has been synthesized by PCR using T. reesei QM 9414 DNAas a template (Schindler et al. (1993) Characterization of the pyruvatekinase-encoding gene (pkil) of Trichoderma reesei. Gene 130: 271.).

cbh2 terminator: The cbh2 terminator sequence starts immediately afterthe STOP codon of the cbh2 gene (to the PvuII site 0.5 kb from the STOPcodon; Mach et al. (1994) Transformation of Trichoderma reesei based onhygromycin B resistance using homologous expression signals. Curr.Genet. 25: 567.) and originates from plasmid pRLM_(ex)30.

egl1 5′-fagment: The 1.8 kb egl1 5′-fragment (ScaI - StuI) has beenisolated from T. reesei QM 6a (Mandels and Reese (1957) Induction ofcellulase in Trichoderma viridae as influenced by carbon sources andmetals. J. Bacteriol. 73: 269.). This fragment is situated about 1.35 kbupstream from the egl1 coding region and it was used to target theintegration of the the transforning DNA into the egl1 locus.

egl1 3′-fragment: The 1.6 kb egl1 3′-fragment (ScaI -XhoI) was, like the5′-fragment, isolated from T. reesei QM 6a. The fragment is situated 0.3kb downstream from the end of the egl1 gene and it was used fortargeting of the transforming DNA into the egl1 locus.

The standard DNA methods described by Sambrook et al. (1989) In:Molecular cloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. were used in construction ofthe vectors. The restriction enzymes, T4 DNA ligase, Klenow fagment ofthe DNA polymerase I, T4 DNA polymerase, polynucleotide kinase and Taqpolymerase were from Boelringer Mannheim, Germany) and New EnglandBiolabs (USA). Each enzyme was used according to the supplier'sinstructions. Plasmid DNA was isolated by using Qiagen columns (QiagenGmbH, Germany) or Promega Magic Minipreps (Promega, USA) according tothe manufacturer's protocols. The oligonucleotides used in thePCR-reactions and in sequencing reactions were synthetized by a ABI(Applied Biosystems, USA) 381A DNA Synthetizer. DNA sequencing was doneas described in Example 19.

DNA fragments for cloning or transfornations were isolated fromlow-melting-point agarose gels (FMC Bioproducts, USA) by β-agarase Itreatment (New England Biolabs, USA) or by using the QIAEX GelExtraction Kit (Qiagen GmbH, Germany) according to the supplier'sinstructions.

T. reesei ALKO3620 was transformed as described by Penttilä et al.(1987) A versatile tansformation system for the cellulolytic filamentousfungus Trichoderma reesei. Gene 61: 155.) with the modificationsdescribed in Karhunen et al. (1993) High frequency one-step genereplacement in Trichoderma reesei. I Endoglucanase I overproduction.Mol. Gen. Genet. 241: 515.). T. reesei transformants were transferred ona selective medium and purified through conidia. Transformants werestabilized by growing them on selective slants for two generations priorto sporulating on potato dextrose agar.

Example 30

Characteristics of the Melanocarpus albomyces ALKO4237 20K-cellulaseproducing transformants

The purified transformants were grown in shake flasks in a mediumcontaining 4% whey, 1.5% complex nitrogen source derived from grain, 5%KH₂PO₄and 0.5% (NH₄)₂SO₄. Cultures were grown at 30° C. and 250 rpm for7 days.

The culture supernatants were blotted directly onto nitrocellulosefilters by a dot-blot apparatus. CBHI was detected by immunostainingusing a CBHI specific monoclonal antibody CI-258 and EGI by spesificmonoclonal antibody EI-2 (Aho et al. (1991) Monoclonal antibodiesagainst core and cellulose-binding domains of Trichoderma reeseicellobiohydrolases I and II and endoglucanase I. Eur. J. Biochem. 200:643.) and the ProtoBlot Western blot AP system (Promega. USA) accordingto the recommondations of the manufacturer.

The T. reesei strains ALKO3620/pALK1231/14, ALKO3620/pALK1231/16,ALKO3620/pALK1231/20 and ALKO3620/pALK1231/59 do not contain the cbh1gene. The cbh1 gene is replaced by the amdS marker gene and the20K-cellulase construct in pALK1231 expression cassette. The chh1 genereplacement was verified in Southern hybridisations. The T. reeseistrains ALKO3620/pALK1235/40 and ALKO3620/pALK1235/49 do not contain theegl1 gene. The egl1 gene is replaced by the hph marker gene and the20K-cellulase construct in pALK1235 expression cassettes. The egl1 genereplacement was verified in Southern hybridisations. The host strainALKO3620 used in the transformations is deficient of the egl2 gene(replaced by ble gene from Streptoalloteichus hindustanus (Mattern etal., 1988, Drocourt et al., 1990). Thus the strans do not produceTrichoderma's cellulase components EGII and CBHI or EGI.

Samples from the culture supernatants were run on polyacrylanide slabgels containing 0.1% SDS on Bio-Rad Mini Protean II electrophoresissystem (USA). The polyclonal antibody prepared against the purified20K-cellulase was used to detect the produced protein in Western blots.In the detection, Promega's ProtoBlot® AP System was used. The Westernresult is shown in FIG. 30. The transfornants ALKO3620/pALK1235/49,ALKO3620/pALK1 235/40, ALKO3620/pALK1231/14 and ALKO3620/pALK1231/16(lanes 1, 2,4 and 5) produce a protein which reacts with the polyclonal20K-cellulase antiserum. The size of the protein produced bytransformants is same as the size of purified 20K-cellulase (lane 6).ALKO3620 (lane 3) does not produce corresponding protein.

The endoglucanase activities of the transformants were determined asdescribed in Example 10. When 2% carboxymethylcellulose (CMC) was usedas a substrate reaction temperature was lifted up to 70 ° C. and thusthe endoglucanase activity of ALKO3620 was heat inactivated. When using1% hydroxyethylcellulose as a substrate heat inactivation was performedbefore enzymatic activity measurements. Samples from growth medium werediluted to 0.05 M HEPES, pH 7.0-buffer and incubated 20 min in 70° C.Heat inactivation of endoglucanase I (the major endoglucanase left inALKO3620) was almost complete. The activity of egl1-negativetransformants dropped about 30% in heat inactivation which indicates theminor heat inactivation of 20K-cellulase. The endoglucanase activitiesare presented in Table XX. When HEC was the substrate, the 20K-cellulaseactivity was extrapolated to the activity before the heat treatment bydividing the activity obtained after the heat treatrnent with 0.7.

TABLE XX The endoglucanase activities of T. reesei transformantsproducing Melanocarpus albomyces 20K-cellulase. 20K-cellulase activity(artificial units/ml) CMC HEC Substrate 70° C., pH 7.0 50° C., pH 7.0ALKO4237   —*  100** ALKO3620    50***   38*** ALKO3620/pALK1231/14 2400350 ALKO3620/pALK1231/16 2600 350 ALKO3620/pALK1231/20 6500 750ALKO3620/pALK1231/59 6800 750 ALKO3620/pALK1235/40 2400 325ALKO3620/pALK1235/49 2100 350 *not measured **not heat inactivated,contains also 50K-cellulase, 50K-cellulase B and other cellulaseactivities ***activity due to Trichoderma cellulases

The endoglucanase activities of the T. reesei host stain ALKO3620 arealmost totally heat inactivated at 70° C. Melanocarpus albomyces20K-cellulase producing transformants produce substantial amounts ofrelative heat stable 20K-cellulase. The endoglucanase production levelof transformants is several times higher than that of 20K-cellulaseparental strain ALKO04237.

Example 31

Production of Melanocarpus albonyces ALKO4237 50K-cellulase in T. reesei

The Trichoderma reesei strains were constructed for Melanocarpusalbomyces ALKO4237 50K-cellulase production. Strains produceMelanocarpus 50K-cellulase and are unable to produce T. reesei'sendoglucanase II and cellobiohydrolase I or endoglucanase I. Inconstruction of the Melanocarpus albomyces 50K-cellulase producingstrains, the parental Trichoderma reesei strain ALKO3620 was transformedwith the expression cassettes from the plasmid pALK1238 or pALK1240(FIGS. 31 and 32). In the cassettes 50K-cellulase is expressed from thestrong cbh1 promoter. The integration of the expression cassettesresults in the replacements of the parental cbh1 (pALK1238) or the egl1(pALK1240) genes. Cloning and transformation were done as described inExample 29, except that 20K-cellulase gene was replaced by 50K-cellulasegene (1.7 kb fragment beginning from ATG-codon) described in Example 22.The Melanocarpus albomyces 50K-cellulase producing transformants arethen characterized similar to example 30 with modifications obvious to aperson skilled in the art. The Melanocarpus albomyces 50K-cellulase Band protein-with-CBD producing transformants can be created similar toExamples 29 and 30 with modifications obvious to a person skilled in theart.

Having now fully described the invention, it will be understood by thosewith skill in the art that the invention may be performed within a wideand equivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof. All references cited herein are fully incorporated herein byreference.

45 30 amino acids amino acid linear peptide Melanocarpus albomycesALKO4237 Peptide 1..30 /label= No_429 1 Ala Asn Gly Gln Ser Thr Arg TyrTrp Asp Cys Cys Lys Pro Ser Cys 1 5 10 15 Gly Trp Arg Gly Lys Gly ProVal Asn Gln Pro Val Tyr Ser 20 25 30 7 amino acids amino acid linearpeptide Melanocarpus albomyces ALKO4237 Peptide 1..7 /label= No_430 2Tyr Gly Gly Ile Ser Ser Arg 1 5 4 amino acids amino acid linear peptideMelanocarpus albomyces ALKO4237 Peptide 1..4 /label= No_431 3 Cys GlyTrp Arg 1 6 amino acids amino acid linear peptide Melanocarpus albomycesALKO4237 Peptide 1..6 /label= No_432 4 Pro Ser Cys Gly Trp Arg 1 5 6amino acids amino acid linear peptide Melanocarpus albomyces ALKO4237Peptide 1..6 /label= No_433 5 Tyr Trp Asp Cys Cys Lys 1 5 17 amino acidsamino acid linear peptide Melanocarpus albomyces ALKO4237 Peptide 1..17/label= No_439 6 Gln Glu Cys Asp Ser Phe Pro Glu Pro Leu Lys Pro Gly CysGln Trp 1 5 10 15 Arg 8 amino acids amino acid linear peptideMelanocarpus albomyces ALKO4237 Peptide 1..8 /label= fr9 7 Arg His AspAsp Gly Gly Phe Ala 1 5 7 amino acids amino acid linear peptideMelanocarpus albomyces ALKO4237 Peptide 1..7 /label= fr14 8 Tyr Trp AspCys Cys Lys Pro 1 5 18 amino acids amino acid linear peptideMelanocarpus albomyces ALKO4237 Peptide 1..18 /label= fr16 9 Gly Lys GlyPro Val Asn Gln Pro Val Tyr Ser Cys Asp Ala Asn Phe 1 5 10 15 Gln Arg 10amino acids amino acid linear peptide Melanocarpus albomyces ALKO4237Peptide 1..10 /label= fr17 10 Val Gln Cys Pro Glu Glu Leu Val Ala Arg 15 10 15 amino acids amino acid linear peptide Melanocarpus albomycesALKO4237 Peptide 1..15 /label= fr28 11 Asp Trp Phe Gln Asn Ala Asp AsnPro Ser Phe Thr Phe Glu Arg 1 5 10 15 30 amino acids amino acid linearpeptide Melanocarpus albomyces ALKO4237 Peptide 1..30 /label= fr30 12Thr Met Val Val Gln Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn 1 5 1015 His Phe Asp Leu Asn Ile Pro Gly Gly Gly Val Gly Leu Phe 20 25 30 10amino acids amino acid linear peptide Melanocarpus albomyces ALKO4237Peptide 1..10 /label= No_507 13 Val Tyr Leu Leu Asp Glu Thr Glu His Arg1 5 10 12 amino acids amino acid linear peptide Melanocarpus albomycesALKO4237 Peptide 1..12 /label= No_509 14 Xaa Xaa Leu Asn Pro Gly Gly AlaTyr Tyr Gly Thr 1 5 10 17 amino acids amino acid linear peptideMelanocarpus albomyces ALKO4237 Peptide 1..17 /label= No_563 15 Met SerGlu Gly Ala Glu Cys Glu Tyr Asp Gly Val Cys Asp Lys Asp 1 5 10 15 Gly 14amino acids amino acid linear peptide Melanocarpus albomyces ALKO4237Peptide 1..14 /label= No_565 16 Asn Pro Tyr Arg Val Xaa Ile Thr Asp TyrTyr Gly Asn Ser 1 5 10 24 amino acids amino acid linear peptideMelanocarpus albomyces ALKO4237 Peptide 1..24 /label= No_603 17 Asp ProThr Gly Ala Arg Ser Glu Leu Asn Pro Gly Gly Ala Tyr Tyr 1 5 10 15 GlyThr Gly Tyr Xaa Asp Ala Gln 20 13 amino acids amino acid linear peptideMelanocarpus albomyces ALKO4237 Peptide 1..13 /label= No_605 18 Xaa XaaVal Pro Asp Tyr His Gln His Gly Val Asp Ala 1 5 10 12 amino acids aminoacid linear peptide Melanocarpus albomyces ALKO4237 Peptide 1..12/label= No_610 19 Asn Glu Met Asp Ile Xaa Glu Ala Asn Ser Arg Ala 1 5 1025 amino acids amino acid linear peptide Melanocarpus albomyces ALKO4237Peptide 1..25 /label= No_611 20 Leu Pro Xaa Gly Met Asn Ser Ala Leu TyrLeu Ser Glu Met Asp Pro 1 5 10 15 Thr Gly Ala Arg Ser Glu Leu Asn Pro 2025 21 amino acids amino acid linear peptide Melanocarpus albomycesALKO4237 Peptide 1..21 /label= No_612 21 Val Glu Pro Ser Pro Glu Val ThrTyr Ser Asn Leu Arg Xaa Gly Glu 1 5 10 15 Ile Xaa Gly Xaa Phe 20 19amino acids amino acid linear peptide Melanocarpus albomyces ALKO4237Peptide 1..19 /label= No_619 22 Asp Gly Cys Gly Trp Asn Pro Tyr Arg ValVal Ile Thr Thr Asp Tyr 1 5 10 15 Tyr Asn Asn 10 amino acids amino acidlinear peptide Melanocarpus albomyces ALKO4237 Peptide 1..10 /label=No_620 23 Leu Pro Cys Gly Met Xaa Ser Ala Leu Tyr 1 5 10 22 amino acidsamino acid linear peptide Melanocarpus albomyces ALKO4237 Peptide 1..22/label= No_621 24 Ala Asp Gly Cys Gln Pro Arg Thr Asn Tyr Ile Val LeuAsp Asp Leu 1 5 10 15 Leu His Pro Xaa Xaa Gln 20 9 amino acids aminoacid linear peptide Melanocarpus albomyces ALKO4237 Peptide 1..9 /label=No_534 25 Val Gly Asn Pro Asp Phe Tyr Gly Lys 1 5 8 amino acids aminoacid linear peptide Melanocarpus albomyces ALKO4237 Peptide 1..8 /label=No_535 26 Phe Gly Pro Ile Gly Ser Thr Tyr 1 5 12 amino acids amino acidlinear peptide Melanocarpus albomyces ALKO4237 Peptide 1..12 /label=No_631 27 Leu Ser Gln Tyr Phe Ile Gln Asp Gly Glu Arg Lys 1 5 10 11amino acids amino acid linear peptide Melanocarpus albomyces ALKO4237Peptide 1..11 /label= No_632 28 Phe Thr Val Val Ser Arg Phe Glu Glu AsnLys 1 5 10 19 amino acids amino acid linear peptide Melanocarpusalbomyces ALKO4237 Peptide 1..19 /label= No_636 29 His Glu Tyr Gly ThrAsn Val Gly Ser Arg Phe Tyr Leu Met Asn Gly 1 5 10 15 Pro Asp Lys 936base pairs nucleic acid single linear DNA (genomic) Melanocarpusalbomyces ALKO4237 exon 33..115 /codon_start= 33 /product=“20K-cellulase” exon 187..435 /product= “20K-cellulase” exon 506..881/product= “20K-cellulase” 30 TCGCCCCTAA CCGAGAACCA AAGACTCCAA GAATGCGCTCTACTCCCGTT CTCCGCGCCC 60 TCCTGGCCGC AGCATTGCCC CTCGGGGCCC TCGCCGCCAACGGTCAGTCC ACGAGGTAAC 120 TGATCACCCG CCTCATTACG CGTGCCGACC GGACCGCGTTCAGGGCTCAC TGCTCACCGC 180 ATCCAGATAC TGGGACTGCT GCAAGCCGTC GTGCGGCTGGCGCGGAAAGG GCCCCGTGAA 240 CCAGCCCGTC TACTCGTGCG ACGCCAACTT CCAGCGCATCCACGACTTCG ATGCCGTCTC 300 GGGCTGCGAG GGCGGCCCCG CCTTCTCGTG CGCCGACCACAGCCCCTGGG CCATTAATGA 360 CAACCTCTCG TACGGCTTCG CGGCGACTGC ACTCAGCGGCCAGACCGAGG AGTCGTGGTG 420 CTGTGCCTGC TACGCGTGAG TGTGCTTGGG CCCAACGTCGGTGATTCCGG AGTTCAGACC 480 ACTGACCCAG CGACCCGCTC GCCAGTCTGA CCTTTACATCGGGTCCCGTG GCCGGCAAGA 540 CCATGGTCGT CCAGTCGACC AGCACGGGCG GCGACCTCGGCAGCAACCAC TTCGACCTCA 600 ACATCCCCGG CGGCGGCGTC GGCCTCTTCG ACGGCTGCACTCCCCAGTTC GGCGGCCTCC 660 CGGGCGCACG GTACGGCGGC ATCTCGTCGC GCCAGGAGTGCGACTCGTTC CCCGAGCCGC 720 TCAAGCCCGG CTGCCAGTGG CGCTTCGACT GGTTCCAGAACGCCGACAAC CCGTCCTTTA 780 CCTTCGAGCG GGTCCAGTGC CCCGAGGAGC TGGTCGCTCGGACCGGCTGC AGGCGCCACG 840 ACGACGGCGG CTTCGCCGTC TTCAAGGCCC CCAGCGCCTGATCCGTTTTT GGGCAGTGTC 900 CGTGTGACGG CAGCTACGTG GAACGACCTG GAGCTC 936235 amino acids amino acid linear protein Melanocarpus albomycesALKO4237 Protein 1..235 /label= 20K-cellulase 31 Met Arg Ser Thr Pro ValLeu Arg Ala Leu Leu Ala Ala Ala Leu Pro 1 5 10 15 Leu Gly Ala Leu AlaAla Asn Gly Gln Ser Thr Arg Tyr Trp Asp Cys 20 25 30 Cys Lys Pro Ser CysGly Trp Arg Gly Lys Gly Pro Val Asn Gln Pro 35 40 45 Val Tyr Ser Cys AspAla Asn Phe Gln Arg Ile His Asp Phe Asp Ala 50 55 60 Val Ser Gly Cys GluGly Gly Pro Ala Phe Ser Cys Ala Asp His Ser 65 70 75 80 Pro Trp Ala IleAsn Asp Asn Leu Ser Tyr Gly Phe Ala Ala Thr Ala 85 90 95 Leu Ser Gly GlnThr Glu Glu Ser Trp Cys Cys Ala Cys Tyr Ala Leu 100 105 110 Thr Phe ThrSer Gly Pro Val Ala Gly Lys Thr Met Val Val Gln Ser 115 120 125 Thr SerThr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn Ile 130 135 140 ProGly Gly Gly Val Gly Leu Phe Asp Gly Cys Thr Pro Gln Phe Gly 145 150 155160 Gly Leu Pro Gly Ala Arg Tyr Gly Gly Ile Ser Ser Arg Gln Glu Cys 165170 175 Asp Ser Phe Pro Glu Pro Leu Lys Pro Gly Cys Gln Trp Arg Phe Asp180 185 190 Trp Phe Gln Asn Ala Asp Asn Pro Ser Phe Thr Phe Glu Arg ValGln 195 200 205 Cys Pro Glu Glu Leu Val Ala Arg Thr Gly Cys Arg Arg HisAsp Asp 210 215 220 Gly Gly Phe Ala Val Phe Lys Ala Pro Ser Ala 225 230235 1894 base pairs nucleic acid single linear DNA (genomic)Melanocarpus albomyces ALKO4237 exon 233..838 /product= “50K-cellulase”exon 916..1596 /product= “50K-cellulase” 32 GAATTCGGGG GTTGCCAGGGAGTCGTACAG GGGTGGGTGG AGGGGGATGG GGGATGGAAG 60 GGGGATGGAG AAGAAAGCATATATGGGACG TTTGTGCTCG CCGGCTCCCC TCTGCCACGT 120 TCCCTTGCCT CCTTGCCTGGGTTGTTGTTG GTCTTCCCTT CACCATCCGA CAAACCAACC 180 TGCTGCGGGT GAACTCGCAGAGCGCCTTCG GACGACGACA GACAGACGCA CCATGACTCG 240 CAACATCGCC CTGCTCGGCGCCGCGTCGGC GCTCCTGGGC CTCGCCCACG GCCAGAAGCC 300 GGGCGAGACG CCCGAGGTGCACCCGCAGCT GACGACGTTC CGGTGCACCA AGGCGGACGG 360 GTGCCAGCCG CGGACCAACTACATTGTGCT GGACTCGCTG TCGCACCCGG TGCACCAGGT 420 GGACAACGAC TACAACTGCGGCGACTGGGG GCAGAAGCCC AACGCGACGG CGTGCCCGGA 480 CGTCGAGTCG TGCGCGCGCAACTGCATCAT GGAGGGCGTG CCCGACTACA GCCAGCACGG 540 CGTCACGACG AGCGACACGTCGCTGCGCCT GCAGCAGCTC GTCGACGGCC GCCTCGTCAC 600 GCCGCGCGTC TACCTGCTCGACGAGACCGA GCACCGCTAC GAGATGATGC ACCTGACCGG 660 CCAGGAGTTC ACCTTTGAGGTCGACGCCAC CAAGCTGCCC TGCGGCATGA ACAGCGCCCT 720 CTACCTGTCC GAGATGGACCCGACCGGCGC CCGGAGCGAG CTCAACCCCG GCGGTGCCTA 780 CTACGGCACC GGCTACTGCGACGCCCAGTG CTTCGTGACG CCATTCATCA ACGGCATTGT 840 GAGTGTTCCC CTTTGGCCCCCCCCCTGAAA ATAGATGTAC CTGGGTGCTA ACCCCGGGGT 900 GTCGCACCAA AACAGGGCAACATCGAGGGC AAGGGCTCGT GCTGCAACGA GATGGACATC 960 TGGGAGGCCA ACTCGCGGGCGACGCACGTG GCGCCGCACA CGTGCAACCA GACGGGTCTG 1020 TACATGTGCG AGGGCGCCGAGTGCGAGTAC GACGGCGTGT GCGACAAGGA CGGGTGCGGG 1080 TGGAACCCGT ACCGGGTCAACATCACCGAC TACTACGGCA ACTCGGACGC GTTCCGCGTC 1140 GACACGCGGC GGCCCTTCACCGTGGTGACG CAGTTCCCGG CCGACGCCGA GGGCCGGCTC 1200 GAGAGCATCC ACCGGCTGTACGTGCAGGAC GGCAAGGTGA TCGAGTCGTA CGTCGTCGAC 1260 GCGCCGGGCC TGCCCCGGACCGACTCGCTC AACGACGAGT TCTGCGCCGC CACGGGCGCC 1320 GCGCGCTACC TCGACCTCGGCGGCACCGCG GGCATGGGCG ACGCCATGAC GCGCGGCATG 1380 GTGCTGGCCA TGAGCATCTGGTGGGACGAG TCCGGCTTCA TGAACTGGCT CGACAGCGGC 1440 GAGGCCGGCC CCTGCCTGCCCGACGAGGGC GACCCCAAGA ACATTGTCAA GGTCGAGCCC 1500 AGCCCCGAGG TCACCTACAGCAACCTGCGC TGGGGCGAGA TCGGGTCGAC CTTTGAGGCC 1560 GAGTCCGACG ACGACGGCGACGGCGACGAC TGCTAGATAA CTAACTAGTG GGCGGAAAGG 1620 GCGGGGGATG CGTAACTTACATACAGCCCG GAGTTGTTTT GAGTGTAGAG TATTGAGCTT 1680 TCGATGTGTT AGTTGAGTGGAATGGAAAAT TCGCGTCTTT GCCCCGGTGG TTGCGATAAA 1740 CAATAGTCGG CTGGTGCATTTGTGACACTT CAATTGCGCT GTTGGCTTGG TGACAGACAC 1800 GGCAGCGTCG ATGACCCGACACCCAGAATA ATTCGCATGG TTGATTATGT TATTGTGCTT 1860 TAAATCGGAG GCTGATGCTCATCTCTTCGA ATTC 1894 428 amino acids amino acid linear proteinMelanocarpus albomyces ALKO4237 Protein 1..428 /label= 50K-cellulase 33Met Thr Arg Asn Ile Ala Leu Leu Gly Ala Ala Ser Ala Leu Leu Gly 1 5 1015 Leu Ala His Gly Gln Lys Pro Gly Glu Thr Pro Glu Val His Pro Gln 20 2530 Leu Thr Thr Phe Arg Cys Thr Lys Ala Asp Gly Cys Gln Pro Arg Thr 35 4045 Asn Tyr Ile Val Leu Asp Ser Leu Ser His Pro Val His Gln Val Asp 50 5560 Asn Asp Tyr Asn Cys Gly Asp Trp Gly Gln Lys Pro Asn Ala Thr Ala 65 7075 80 Cys Pro Asp Val Glu Ser Cys Ala Arg Asn Cys Ile Met Glu Gly Val 8590 95 Pro Asp Tyr Ser Gln His Gly Val Thr Thr Ser Asp Thr Ser Leu Arg100 105 110 Leu Gln Gln Leu Val Asp Gly Arg Leu Val Thr Pro Arg Val TyrLeu 115 120 125 Leu Asp Glu Thr Glu His Arg Tyr Glu Met Met His Leu ThrGly Gln 130 135 140 Glu Phe Thr Phe Glu Val Asp Ala Thr Lys Leu Pro CysGly Met Asn 145 150 155 160 Ser Ala Leu Tyr Leu Ser Glu Met Asp Pro ThrGly Ala Arg Ser Glu 165 170 175 Leu Asn Pro Gly Gly Ala Tyr Tyr Gly ThrGly Tyr Cys Asp Ala Gln 180 185 190 Cys Phe Val Thr Pro Phe Ile Asn GlyIle Gly Asn Ile Glu Gly Lys 195 200 205 Gly Ser Cys Cys Asn Glu Met AspIle Trp Glu Ala Asn Ser Arg Ala 210 215 220 Thr His Val Ala Pro His ThrCys Asn Gln Thr Gly Leu Tyr Met Cys 225 230 235 240 Glu Gly Ala Glu CysGlu Tyr Asp Gly Val Cys Asp Lys Asp Gly Cys 245 250 255 Gly Trp Asn ProTyr Arg Val Asn Ile Thr Asp Tyr Tyr Gly Asn Ser 260 265 270 Asp Ala PheArg Val Asp Thr Arg Arg Pro Phe Thr Val Val Thr Gln 275 280 285 Phe ProAla Asp Ala Glu Gly Arg Leu Glu Ser Ile His Arg Leu Tyr 290 295 300 ValGln Asp Gly Lys Val Ile Glu Ser Tyr Val Val Asp Ala Pro Gly 305 310 315320 Leu Pro Arg Thr Asp Ser Leu Asn Asp Glu Phe Cys Ala Ala Thr Gly 325330 335 Ala Ala Arg Tyr Leu Asp Leu Gly Gly Thr Ala Gly Met Gly Asp Ala340 345 350 Met Thr Arg Gly Met Val Leu Ala Met Ser Ile Trp Trp Asp GluSer 355 360 365 Gly Phe Met Asn Trp Leu Asp Ser Gly Glu Ala Gly Pro CysLeu Pro 370 375 380 Asp Glu Gly Asp Pro Lys Asn Ile Val Lys Val Glu ProSer Pro Glu 385 390 395 400 Val Thr Tyr Ser Asn Leu Arg Trp Gly Glu IleGly Ser Thr Phe Glu 405 410 415 Ala Glu Ser Asp Asp Asp Gly Asp Gly AspAsp Cys 420 425 2000 base pairs nucleic acid single linear DNA (genomic)Melanocarpus albomyces ALKO4237 exon 154..729 /product= “50K-cellulaseB” exon 810..946 /product= “50K-cellulase B” exon 1018..1230 /product=“50K-cellulase B” exon 1308..1551 /product= “50K-cellulase B” exon1637..1767 /product= “50K-cellulase B” exon 1831..1888 /product=“50K-cellulase B” 34 CCCGGTCTGG AGACGGGGAG CGCGCCAGCG ACGCAGGATAAGAAGGCGAC GACCGCGCCT 60 CCGAGCCAGG CCCAGGACAG CAGGAGAACT CGCCACGCGCAAGCAGCACG CCCGATCGAC 120 AGTGTCCCGC TCTGCCCACA GCACTCTGCA ACCATGATGATGAAGCAGTA CCTCCAGTAC 180 CTCGCGGCCG CGCTGCCGCT CGTCGGCCTC GCCGCCGGCCAGCGCGCTGG TAACGAGACG 240 CCCGAGAACC ACCCCCCGCT CACCTGGCAG AGGTGCACGGCCCCGGGCAA CTGCCAGACC 300 GTGAACGCCG AGGTCGTCAT TGACGCCAAC TGGCGCTGGCTGCACGACGA CAACATGCAG 360 AACTGCTACG ACGGCAACCA GTGGACCAAC GCCTGCAGCACCGCCACCGA CTGCGCTGAG 420 AAGTGCATGA TCGAGGGTGC CGGCGACTAC CTGGGCACCTACGGCGCCTC GACCAGCGGC 480 GACGCCCTGA CGCTCAAGTT CGTCACCAAG CACGAGTACGGCACCAACGT CGGCTCGCGC 540 TTCTACCTCA TGAACGGCCC GGACAAGTAC CAGATGTTCAACCTCATGGG CAACGAGCTT 600 GCCTTTGACG TCGACCTCTC GACCGTCGAG TGCGGCATCAACAGCGCCCT GTACTTCGTC 660 GCCATGGAGG AGGACGGCGG CATGGCCAGC TACCCGAGCAACCAGGCCGG CGCCCGGTAC 720 GGCACTGGGG TGAGTTGAGC TCCGCTTTGT TTCGAGTCGCAACGAGGCAC TTTCTGGGCG 780 CCGGCTAACT CTCTCGATTC CTCCGACAGT ACTGCGATGCCCAATGCGCT CGTGATCTCA 840 AGTTCGTTGG CGGCAAGGCC AACATTGAGG GCTGGAAGTCGTCCACCAGC GACCCCAACG 900 CTGGCGTCGG CCCGTACGGC AGCTGCTGCG CTGAGATCGACGTCTGGTGA GTGCGAGACC 960 GTCCACCCAG GTTCGGATGC GGGGTGGAAA TTTCGCGGCTAACGGAGCAC CCCCCAGGGA 1020 GTCGAATGCC TATGCCTTCG CTTTCACGCC GCACGCGTGCACGACCAACG AGTACCACGT 1080 CTGCGAGACC ACCAACTGCG GTGGCACCTA CTCGGAGGACCGCTTCGCCG GCAAGTGCGA 1140 CGCCAACGGC TGCGACTACA ACCCCTACCG CATGGGCAACCCCGACTTCT ACGGCAAGGG 1200 CAAGACGCTC GACACCAGCC GCAAGTTCAC GTGCGTGACCCCTTGTGGCG CAACCTTTCT 1260 CTGCCTGCCT GGACACACTG AAACTGACAC GTCGTTTTCGGCTGCAGCGT CGTCTCCCGC 1320 TTCGAGGAGA ACAAGCTCTC CCAGTACTTC ATCCAGGACGGCCGCAAGAT CGAGATCCCG 1380 CCGCCGACGT GGGAGGGCAT GCCCAACAGC AGCGAGATCACCCCCGAGCT CTGCTCCACC 1440 ATGTTCGATG TGTTCAACGA CCGCAACCGC TTCGAGGAGGTCGGCGGCTT CGAGCAGCTG 1500 AACAACGCCC TCCGGGTTCC CATGGTCCTC GTCATGTCCATCTGGGACGA CGTAAGTACC 1560 CGCCGACCTC CCTAGCCACA CAAGCCGCAT CCGGCGAGGCACGCCATCGC TGCTGCTAAC 1620 ACGAGACCGT TCGTAGCACT ACGCCAACAT GCTCTGGCTCGACTCCATCT ACCCGCCCGA 1680 GAAGGAGGGC CAGCCCGGCG CCGCCCGTGG CGACTGCCCCACGGACTCGG GTGTCCCCGC 1740 CGAGGTCGAG GCTCAGTTCC CCGACGCGTA AGACTTGCCCCCGACCCCAA GCTTCCACTT 1800 CTGGATGCCG AATGCTAACA CGCGAAACAG CCAGGTCGTCTGGTCCAACA TCCGCTTCGG 1860 CCCCATCGGC TCGACCTACG ACTTCTAAGC CGGTCCATGCACTCGCAGCC CTGGGCCCGT 1920 CACGCCCGCC ACCTCCCCTC GCGGAAACTC TCCGTGCGTCGCGGGCTCCA AAGCATTTTG 1980 GCCTCAAGTT TTTTTCGTTC 2000 452 amino acidsamino acid linear protein Melanocarpus albomyces ALKO4237 Protein 1..452/label= 50K-cellulase-B 35 Met Met Met Lys Gln Tyr Leu Gln Tyr Leu AlaAla Ala Leu Pro Leu 1 5 10 15 Val Gly Leu Ala Ala Gly Gln Arg Ala GlyAsn Glu Thr Pro Glu Asn 20 25 30 His Pro Pro Leu Thr Trp Gln Arg Cys ThrAla Pro Gly Asn Cys Gln 35 40 45 Thr Val Asn Ala Glu Val Val Ile Asp AlaAsn Trp Arg Trp Leu His 50 55 60 Asp Asp Asn Met Gln Asn Cys Tyr Asp GlyAsn Gln Trp Thr Asn Ala 65 70 75 80 Cys Ser Thr Ala Thr Asp Cys Ala GluLys Cys Met Ile Glu Gly Ala 85 90 95 Gly Asp Tyr Leu Gly Thr Tyr Gly AlaSer Thr Ser Gly Asp Ala Leu 100 105 110 Thr Leu Lys Phe Val Thr Lys HisGlu Tyr Gly Thr Asn Val Gly Ser 115 120 125 Arg Phe Tyr Leu Met Asn GlyPro Asp Lys Tyr Gln Met Phe Asn Leu 130 135 140 Met Gly Asn Glu Leu AlaPhe Asp Val Asp Leu Ser Thr Val Glu Cys 145 150 155 160 Gly Ile Asn SerAla Leu Tyr Phe Val Ala Met Glu Glu Asp Gly Gly 165 170 175 Met Ala SerTyr Pro Ser Asn Gln Ala Gly Ala Arg Tyr Gly Thr Gly 180 185 190 Tyr CysAsp Ala Gln Cys Ala Arg Asp Leu Lys Phe Val Gly Gly Lys 195 200 205 AlaAsn Ile Glu Gly Trp Lys Ser Ser Thr Ser Asp Pro Asn Ala Gly 210 215 220Val Gly Pro Tyr Gly Ser Cys Cys Ala Glu Ile Asp Val Trp Glu Ser 225 230235 240 Asn Ala Tyr Ala Phe Ala Phe Thr Pro His Ala Cys Thr Thr Asn Glu245 250 255 Tyr His Val Cys Glu Thr Thr Asn Cys Gly Gly Thr Tyr Ser GluAsp 260 265 270 Arg Phe Ala Gly Lys Cys Asp Ala Asn Gly Cys Asp Tyr AsnPro Tyr 275 280 285 Arg Met Gly Asn Pro Asp Phe Tyr Gly Lys Gly Lys ThrLeu Asp Thr 290 295 300 Ser Arg Lys Phe Thr Val Val Ser Arg Phe Glu GluAsn Lys Leu Ser 305 310 315 320 Gln Tyr Phe Ile Gln Asp Gly Arg Lys IleGlu Ile Pro Pro Pro Thr 325 330 335 Trp Glu Gly Met Pro Asn Ser Ser GluIle Thr Pro Glu Leu Cys Ser 340 345 350 Thr Met Phe Asp Val Phe Asn AspArg Asn Arg Phe Glu Glu Val Gly 355 360 365 Gly Phe Glu Gln Leu Asn AsnAla Leu Arg Val Pro Met Val Leu Val 370 375 380 Met Ser Ile Trp Asp AspHis Tyr Ala Asn Met Leu Trp Leu Asp Ser 385 390 395 400 Ile Tyr Pro ProGlu Lys Glu Gly Gln Pro Gly Ala Ala Arg Gly Asp 405 410 415 Cys Pro ThrAsp Ser Gly Val Pro Ala Glu Val Glu Ala Gln Phe Pro 420 425 430 Asp AlaGln Val Val Trp Ser Asn Ile Arg Phe Gly Pro Ile Gly Ser 435 440 445 ThrTyr Asp Phe 450 887 base pairs nucleic acid single linear DNA (genomic)Melanocarpus albomyces ALKO4237 exon 351..455 /product=“protein-with-CBD” 36 CCATGGACGC GAACTGCGAC GTCTTCTGCC CCGAGCTGAAGACCCAGAGC ATCCAGACCG 60 GCAACCAGTG CACCCAGGAG ATGAAGGTCT ACGAGAACATTGACGGCTGG CTCGACAGCC 120 TGCCCGGCAA CGTCCCCATC ACCGGTCCGC AGCCCGGCTCTGGTAAGTCA AAGAGATGAT 180 GCCTACCTAC CTTCCCACCT TCCCACCCAG CCGCAAATACCTTTCTCCCT CCCCGTGCCC 240 CGTATTCTTT CAACGCCCCG AGACTGACAG ACCCGCTCGTCCCAGGCGGC AACCCCGGCA 300 ACGGCGGCGG CAGCAACCCG GGCAACGGCG GCGGCGGCGGCTGCACCGTC CAGAAGTGGG 360 GCCAGTGCGG CGGCATCGGC TACTCGGGCT GCACCACCTGCAAGGCCGGC TCGACCTGCC 420 CGGCCCAGAA CGAGTACTAC TCGCAGTGCC TGTAAAGCGGCCGTGGGCTA GGTGGCCGAG 480 CGGGGGGGTT TCTTCATTGG TTGAGCAAAT AGAACAGGATTTCCGGCTCG TTGGCAGCGG 540 CGCGCCGCGG GGATGGTGTT GTACAATTCA AGACCTCAGTACCGAGGGAC CTGGAAAGGA 600 GTCAGTCTGC TTGTACGGAG GCTGGCTGCC CCGTGGCGGCGCTGGCAAGG TAGATAGCCC 660 TTCATTGCTG TAACTAGTAT GCTATATACC TCTGCACATTTGCAGCCCCA TGGTGTGAAC 720 AACAAGTGAC AAGGCTTCCA GTTCCAGCCT CGCGCAATTGTCACGATATC CTTGGTCCAT 780 CTATATGTAT GGGCATGAGC GAGTCGAGAA AATGTACCGCGAAAAATCGT AGTGACCTGC 840 GCACTGCGCC GTTCTACCAC CGTAGGATTG AAGTGAATCTCGAATTC 887 34 amino acids amino acid linear peptide Melanocarpusalbomyces ALKO4237 Protein 1..34 /label= prot-with-CBD 37 Gln Lys TrpGly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Cys Thr Thr 1 5 10 15 Cys LysAla Gly Ser Thr Cys Pro Ala Gln Asn Glu Tyr Tyr Ser Gln 20 25 30 Cys Leu29 base pairs nucleic acid single linear cDNA unknown 38 ATAGAATTCTAYTGGGAYTG YTGYAARCC 29 26 base pairs nucleic acid single linear cDNAunknown 39 ATAGAATTCT TRTCNGCRTT YTGRAA 26 17 base pairs nucleic acidsingle linear cDNA unknown 40 GAYGARACNG ARCAYMG 17 17 base pairsnucleic acid single linear cDNA unknown 41 TANGCNCCNC CNGGRTT 17 17 basepairs nucleic acid single linear cDNA unknown 42 AARCAYGART AYGGNAC 1717 base pairs nucleic acid single linear cDNA unknown 43 CCRTARAARTCNGGRTT 17 15 base pairs nucleic acid single linear cDNA unknown 44CCGCGGACTG GCATC 15 16 base pairs nucleic acid single linear cDNAunknown 45 CCGCGGACTG CGCATC 16

We claim:
 1. A nucleic acid molecule encoding a polypeptide having theenzymatic activity of a cellulase, selected from the group consistingof: (a) nucleic acid molecules encoding a polypeptide comprising theamino acid sequence as depicted in FIGS. 19 (SEQ ID NO:17) or 21 (SEQ IDNO:33); (b) nucleic acid molecules encoding a polypeptide comprising theamino acid sequence as depicted in FIGS. 23 (SEQ ID NO:35) or 27 (SEQ IDNO:37); (c) nucleic acid molecules comprising the coding sequence of thenucleotide sequence as depicted in FIGS. 19 (SEQ ID NO:30) or 21 (SEQ IDNO:32); (d) nucleic acid molecules comprising the coding sequence of thenucleotide sequence as depicted in FIGS. 23 (SEQ ID NO:34) or 27 (SEQ IDNO:36); (e) nucleic acid molecules encoding a.polypeptide comprising theamino acid sequence encoded by the DNA insert contained in DSM 11024,DSM 11012, DSM 11025 or DSM 11014; (f) nucleic acid molecules encoding apolypeptide comprising the amino acid sequence encoded by the DNA insertcontained in DSM 11026, DSM 11011, DSM 11013 or DSM 11027; (g) nucleicacid molecules comprising the coding sequence of the DNA insertcontained in DSM 11024, DSM 11012, DSM 11025 or DSM 11014; (h) nucleicacid molecules comprising the coding sequence of the DNA insertcontained in DSM 11026, DSM 11011, DSM 11013 or DSM 11027; (i) nucleicacid molecules hybridizing under stringent conditions to a molecule ofany one of (a), (c), (e) or (g); (j) nucleic acid molecules the codingsequence of which differs from the coding sequence of a nucleic acidmolecule of any one of (a) to (i) due to the degeneracy of the geneticcode; and (k) nucleic acid molecules encoding a polypeptide havingcellulase activity and having an amino acid sequence which shows atleast 80% identity to a sequence as depicted in FIGS. 19 (SEQ ID NO:31),21 (SEQ ID NO:33), 23 (SEQ ID NO:35), or 27 (SEQ ID NO:37).
 2. Thenucleic acid molecule of claim 1 which is RNA.
 3. The nucleic acidmolecule of claim 1 which is DNA.
 4. The DNA of claim 3 which is genomicDNA or cDNA.
 5. A vector containing a nucleic acid molecule of any oneof claims 1 to
 4. 6. The vector of claim 5, in which the nucleic acidmolecule is operably linked to expression control sequences allowingexpression in prokaryotic or eukaryotic host cells.
 7. A host celltransformed with a nucleic acid molecule of any one of claims 1 to 4 orwith a vector of claim 5 or
 6. 8. The host cell of claim 7 which belongsto filamentous fungi.
 9. The host cell of claims 7 or 8 which belongs tothe genus Trichoderma or Aspergillus.
 10. The host cell of claim 9 whichis Trichoderma reesei.
 11. A process for the production of a polypeptidehaving cellulase activity comprising the steps of culturing the hostcell of any one of claims 7 to 10 and recovering the protein from theculture medium.
 12. An oligonucleotide specifically hybridizing understringent conditions to a nucleic acid molecule of any one of claims 1to
 4. 13. A process for the preparation of a cellulase comprising thesteps of culturing a host cell of any one of claims 7 to 10 and eitherrecovering the polypeptide from the cells or separating the cells fromthe culture medium and obtaining the supernatant.
 14. A nucleic acidmolecule according to claim 1, wherein said nucleic acid moleculeencodes a polypeptide comprising the amino acid sequence as depicted inFIG. 19 (SEQ ID NO:31).
 15. A nucleic acid molecule according to claim1, wherein said nucleic acid molecule encodes a polypeptide comprisingthe amino acid sequence as depicted in FIG. 21 (SEQ ID NO:33).
 16. Anucleic acid molecule according to claim 1, wherein said nucleic acidmolecule encodes a polypeptide comprising the amino acid sequence asdepicted in FIG. 23 (SEQ ID NO:35).
 17. A nucleic acid moleculeaccording to claim 1, wherein said nucleic acid molecule encodes apolypeptide comprising the amino acid sequence as depicted in FIG. 27(SEQ ID NO:37).
 18. A nucleic acid molecule according to claim 1,wherein said nucleic acid molecule comprises the coding sequence of thenucleotide sequence as depicted in FIG. 19 (SEQ ID NO:30).
 19. A nucleicacid molecule according to claim 1, wherein said nucleic acid moleculecomprises the coding sequence of the nucleotide sequence as depicted inFIG. 21 (SEQ ID NO:32).
 20. A nucleic acid molecule according to claim1, wherein said nucleic acid molecule comprises the coding sequence ofthe nucleotide sequence as depicted in FIG. 23 (SEQ ID NO:34).
 21. Anucleic acid molecule according to claim 1, wherein said nucleic acidmolecule comprises the coding sequence of the nucleotide sequence asdepicted in FIG. 27 (SEQ ID NO:36).
 22. A nucleic acid moleculeaccording to claim 1, wherein said nucleic acid molecule encodes apolypeptide comprising the amino acid sequence encoded by the DNA insertcontained in DSM
 11024. 23. A nucleic acid molecule according to claim1, wherein said nucleic acid molecule encodes a polypeptide comprisingthe amino acid sequence encoded by the DNA insert contained in DSM11012.
 24. A nucleic acid molecule according to claim 1, wherein saidnucleic acid molecule encodes a polypeptide comprising the amino acidsequence encoded by the DNA insert contained in DSM
 11025. 25. A nucleicacid molecule according to claim 1, wherein said nucleic acid moleculeencodes a polypeptide comprising the amino acid sequence encoded by theDNA insert contained in DSM
 11014. 26. A nucleic acid molecule accordingto claim 1, wherein said nucleic acid molecule encodes a polypeptidecomprising the amino acid sequence encoded by the DNA insert containedin DSM
 11026. 27. A nucleic acid molecule according to claim 1, whereinsaid nucleic acid molecule encodes a polypeptide comprising the aminoacid sequence encoded by the DNA insert contained in DSM
 11011. 28. Anucleic acid molecule according to claim 1, wherein said nucleic acidmolecule encodes a polypeptide comprising the amino acid sequenceencoded by the DNA insert contained in DSM
 11013. 29. A nucleic acidmolecule according to claim 1, wherein said nucleic acid moleculeencodes a polypeptide comprising the amino acid sequence encoded by theDNA insert contained in DSM
 11027. 30. A nucleic acid molecule accordingto claim 1, wherein said nucleic acid molecule comprises the codingsequence of the DNA insert contained in DSM
 11024. 31. A nucleic acidmolecule according to claim 1, wherein said nucleic acid moleculecomprises the coding sequence of the DNA insert contained in DSM 11012.32. A nucleic acid molecule according to claim 1, wherein said nucleicacid molecule comprises the coding sequence of the DNA insert containedin DSM
 11025. 33. A nucleic acid molecule according to claim 1, whereinsaid nucleic acid molecule comprises the coding sequence of the DNAinsert contained in DSM
 11014. 34. A nucleic acid molecule according toclaim 1, wherein said nucleic acid molecule comprises the codingsequence of the DNA insert contained in DSM
 11026. 35. A nucleic acidmolecule according to claim 1, wherein said nucleic acid moleculecomprises the coding sequence of the DNA insert contained in DSM 11011.36. A nucleic acid molecule according to claim 1, wherein said nucleicacid molecule comprises the coding sequence of the DNA insert containedin DSM
 11013. 37. A nucleic acid molecule according to claim 1, whereinsaid nucleic acid molecule comprises the coding sequence of the DNAinsert contained in DSM
 11027. 38. A nucleic acid molecule thathybridizes under stringent conditions to a nucleic acid molecule of anyone of claims 14, 15, 18, 19, 22, 23 24, 25 or 30-33.
 39. A nucleic acidmolecule wherein the coding sequence of said nucleic acid moleculediffers from the coding sequence of a nucleic acid molecule of any oneof claims 14-37 due to the degeneracy of the genetic code.
 40. A nucleicacid molecule wherein the coding sequence of said nucleic acid moleculediffers from the coding sequence of a nucleic acid molecule of any oneof claim 38 due to the degeneracy of the genetic code.
 41. A nucleicacid molecule according to claim 1, wherein said nucleic acid moleculeencodes a polypeptide having cellulase activity and having an amino acidsequence which shows at least 80% identity to a sequence as depicted inFIG. 19 (SEQ ID NO:31).
 42. A nucleic acid molecule according to claim1, wherein said nucleic acid molecule encodes a polypeptide havingcellulase activity and having an amino acid sequence which shows atleast 80% identity to a sequence as depicted in FIG. 21 (SEQ ID NO:33).43. A nucleic acid molecule according to claim 1, wherein said nucleicacid molecule encodes a polypeptide having cellulase activity and havingan amino acid sequence which shows at least 80% identity to a sequenceas depicted in FIG. 23 (SEQ ID NO:35).
 44. A nucleic acid moleculeaccording to claim 1, wherein said nucleic acid molecule encodes apolypeptide having cellulase activity and having an amino acid sequencewhich shows at least 80% identity to a sequence as depicted in FIG. 27(SEQ ID NO:37).